JP2010520168A - New use of glutaminyl cyclase inhibitors - Google Patents

Abstract

The present invention relates generally to inhibitors of glutaminyl peptide cyclotransferase and its use for the treatment and / or prevention of diseases or disorders selected from the group consisting of inflammatory diseases selected from: a Neurodegenerative diseases such as mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down syndrome, familial British dementia, familial Danish dementia, multiple sclerosis, b. Chronic and acute inflammation such as rheumatoid Arthritis, atherosclerosis, restenosis, pancreatitis, c. Fibrosis such as pulmonary fibrosis, liver fibrosis, renal fibrosis, d. Cancer such as cancer / angioendothelioma proliferation, gastric carcinoma, e. Metabolic disease , Eg hypertension, f., And other inflammatory diseases such as neuropathic pain, graft rejection / graft failure / graft vasculopathy, HIV infection / AIDS, pregnancy toxemia, tuberous sclerosis . Furthermore, the present invention relates to respective diagnostic methods, assays and kits.
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Description

  The present invention generally relates to inhibitors of glutaminyl peptide cyclotransferase, as well as rheumatoid arthritis, atherosclerosis, restenosis, pulmonary fibrosis, liver fibrosis, renal fibrosis, pancreatitis, mild cognitive impairment, Alzheimer's disease, down Neurodegeneration in syndrome, familial British dementia, familial Danish dementia, neuropathic pain, graft rejection / graft failure / graft vascular disorder, hypertension, HIV infection / AIDS, pregnancy toxemia, cancer / Relates to its use for the treatment and / or prevention of diseases or disorders selected from the group consisting of hemangioendothelioma growth, tuberous sclerosis and gastric carcinoma.

  Furthermore, the present invention belongs to diagnostic kits and methods based on the use of glutaminyl cyclase inhibitors.

  Glutaminyl cyclase (QC, EC 2.3.2.5) is an intramolecular cyclization of N-terminal glutamyl residue to pyroglutamic acid (5-oxo-proline, pGlu *) under the release of ammonia, and under the release of water. Catalyze the intramolecular cyclization of N-terminal glutamyl residue to pyroglutamic acid.

  QC was first isolated from the latex of tropical plant papaya in 1963 by Messer (Messer, M. 1963 Nature 4874, 1299). Twenty-four years later, the corresponding enzyme activity was found in the animal pituitary (Busby, WHJ et al., 1987 J Biol Chem 262, 8532-8536; Fischer, WH and Spiess, J., 1987 Proc Natl Acad Sci USA 84, 3628-3632). For mammalian QC, the conversion of Gln to pGlu by QC could be demonstrated for TRH and GnRH precursors (Busby, WHJ et al., 1987 J Biol Chem 262, 8532-8536; Fischer, WH And Spiess, J. 1987 Proc Natl Acad Sci USA 84, 3628-3632). In addition, early localization experiments with QC revealed co-localization with its putative catalytic product in the bovine hypothalamus-pituitary tract further improving the function in suggested peptide hormone maturation (Bockers, TM et al., 1995 J Neuroendocrinol 7, 445-453). In contrast, the physiological function of plant QC is less clear. In the case of enzymes from C. papaya, a role in plant defense against pathogenic microorganisms has been suggested (El Moussaoui, A. et al., 2001 Cell Mol Life Sci 58, 556-570). A putative QC from other plants has recently been identified by sequence comparison (Dahl, S. W. et al., 2000 Protein Expr Purif 20, 27-36). However, the physiological functions of these enzymes are still ambiguous.

  Known QCs from plants and animals showed strict specificity for L-glutamine at the N-terminal position of the substrate, and their kinetic behavior was found to follow the Michaelis-Menten equation (Pohl, T. et al., 1991 Proc Natl Acad Sci USA 88, 10059-10063; Consalvo, AP et al., 1988 Anal Biochem 175, 131-138; Gololobov, MY et al., 1996 Biol Chem Hoppe Seyler 377 395-398). However, a comparison of the primary structure of QC from C. papaya with that of a highly conserved QC from mammals revealed no sequence homology (Dahl, SW et al. (2000) Protein Expr Purif 20, 27-36). Plant QC appears to belong to a new family of enzymes (Dahl, SW et al. (2000) Protein Expr Purif 20, 27-36), whereas mammalian QC is evident against bacterial aminopeptidases. It was found to have sequence homology (Bateman, RC et al., 2001 Biochemistry 40, 11246-11250), leading to the conclusion that plant and animal-derived QCs have different evolutionary origins.

  EP 02 011 349.4 discloses a polynucleotide encoding insect glutaminyl cyclase, as well as the polypeptide encoded thereby. This application further provides a host cell comprising an expression vector comprising a polynucleotide of the invention. Host cells containing the isolated polypeptide and insect QC are useful in methods of screening for agents that reduce glutaminyl cyclase activity. Such drugs are described as being useful as insecticides.

  Chemotactic cytokines (chemokines) are proteins that attract and activate leukocytes and are thought to play a fundamental role in inflammation. Chemokines are divided into four groups that are classified according to the appearance of the N-terminal cysteine residue ("C"-; "CC"-; "CXC"-, and "CX3C" -chemokines). “CXC” -chemokines act preferentially on neutrophils. In contrast, “CC” -chemokines preferentially attract monocytes to the site of inflammation. Monocyte infiltration is considered an important event in a number of disease states (Gerard, C. and Rollins, BJ (2001) Nat. Immunol 2, 108-115; Bhatia, M. et al. (2005). ) Pancreatology. 5, 132-144; Kitamoto, S., Egashira K. and Takeshita, A. (2003) J Pharmacol Sci. 91, 192-196). The MCP family, as a family of chemokines, consists of four members (MCP-1-4), showing preference for attracting monocytes but showing differences in their potential (Luini, W. et al. (1994) Cytokine 6, 28-31; Uguccioni, M. et al. (1995) Eur J Immunol 25, 64-68). The following shows both the MCP-1 to 4 cDNA as well as the amino acid sequence:

  Numerous studies have been published, particularly atherosclerosis (Gu, L. et al. (1998) Mol. Cell 2, 275-281; Gosling, J. et al. (1999) J Clin. Invest 103, 773- 778); rheumatoid arthritis (Gong, JH et al. (1997) J Exp. Med 186, 131-137; Ogata, H. et al. (1997) J Pathol. 182, 106-114); pancreatitis (Bhatia, M. et al. (2005) Am. J Physiol Gastrointest. Liver Physiol 288, G1259-G1265); Alzheimer's disease (Yamamoto M. et al. (2005) Am. J Pathol. 166, 1475-1485); (Inoshima, I. et al. (2004) Am. J Physiol Lung Cell Mol. Physiol 286, L1038-L1044); Renal fibrosis (Wada, T. et al. (2004) J Am. Soc. Nephrol. 15, 940-948), and the critical role of MCP-1 in the development of transplant rejection (Saiura, A. et al. (2004) Arterioscler. Thromb. Vasc. Biol. 24 1886-1890). Furthermore, MCP-1 is also used in pregnancy toxemia (Katabuchi, H. et al. (2003) Med Electron Microsc. 36, 253-262), and tumor onset (Ohta, M. et al. (2003) Int. Oncol. 22, 773-778; Li, S. et al. (2005) J Exp. Med 202, 617-624), neuropathic pain (White, FA et al. (2005) Proc. Natl. Acad. Sci. USA), and as a paracrine factor in AIDS (Park, IW, Wang, JF and Groopman, JE (2001) Blood 97, 352-358; Coll, B. et al. (2006) Cytokine 34, 51-55). , May have a role.

  The mature form of human and rodent MCP-1 is post-translationally modified by glutaminyl cyclase (QC) to have an N-terminal pyroglutamyl (pGlu) residue. Since the chemotaxis performance of MCP-1 is mediated by its N-terminus (Van Damme, J. et al. (1999) Chem Immunol 72, 42-56), the N-terminal pGlu modification is N-terminal by aminopeptidase. This creates protein resistance to degradation, which is important. Artificial elongation or degradation causes loss of function, but MCP-1 still binds to its receptor (CCR2) (Proost, P. et al. (1998) J Immunol 160, 4034-4041; Zhang, YJ et al., 1994, J Biol. Chem 269, 15918-15924; Masure, S. et al., 1995 J Interferon Cytokine Res. 15, 955-963; Hemmerich, S. et al. (1999) Biochemistry. 38, 13013-13025).

  Because of the major role of MCP-1 in many disease states, anti-MCP-1 strategies are required. Therefore, small orally available compounds that inhibit the action of MCP-1 are promising candidates for drug development. Inhibitors of glutaminyl cyclase are small orally available compounds that target key steps of pGlu-formation at the N-terminus of MCP-1. (Cynis, H. et al. (2006) Biochim. Biophys. Acta 1764, 1618-1625; Buchholz, M. et al. (2006) J Med Chem 49, 664-677). As a result, the N-terminus of MCP-1 resulting from QC inhibition is not protected by pGlu-residues. Instead, the N-terminus is a glutamine-proline motif that is abundant on the endothelium and in the blood circulation that is prone to cleavage by dipeptidyl peptidases such as dipeptidyl peptidase 4, and fibroblast activation protein (FAP, Seprase) Have This cleavage results in the formation of MCP-1 with the N-terminus truncated. These molecules then develop antagonism at the CCR2 receptor, thus effectively inhibiting the disease state associated with monocytes.

  Atherosclerotic lesions that restrict or prevent coronary blood flow are a major cause of mortality associated with ischemic heart disease, resulting in deaths of 500,000 to 600,000 people each year. Percutaneous coronary angioplasty (PTCA) to open occluded arteries was performed in 1996 in over 550,000 patients in the United States and 945,000+ patients worldwide (Lemaitre et al., 1996). The main limitation of this technique is the problem of vascular closure after PTCA, both immediately after PTCA (acute occlusion) and long-term (restenosis): 30% partially lesioned patients and 50% chronic Totally lesioned patients will progress to restenosis after angioplasty. In addition, restenosis is a significant problem in patients undergoing saphenous vein bypass grafts. A number of factors appear to be involved in the mechanism of acute occlusion and blood vessels with resulting arterial closure and / or platelet deposition along the damaged length of newly opened blood vessels It can be caused by recoil and subsequent fibrin / red blood cell thrombus formation.

  Restenosis after angioplasty is a slower process, accompanied by the release of cell-derived growth factor from adherent platelets, and subsequent inflammatory cells that contribute to intimal smooth muscle cell proliferation and vascular hyperplasia Involved in the early, but not critical, formation of thrombosis with local infiltration. It is important to note that multiple processes of these thrombosis, cell proliferation, cell migration, and inflammation each seem to contribute to the restenosis process.

  In the United States, a 30-50% restenosis rate is interpreted as 120,000 to 200,000 US patients at risk from restenosis. Only 80% of these patients choose repeated angioplasty (the remaining 20% choose coronary artery bypass grafting), which adds to the cost of coronary artery bypass grafting for the remaining 20% And the total cost of restenosis treatment can easily amount to billions of dollars in the United States. Thus, successful prevention of restenosis can result in significant health care savings as well as significant therapeutic benefits.

Monocyte chemoattractant protein 1 (MCP-1, CCL2) is a family of potent chemotactic cytokines (CC chemokines) that regulate the transport of leukocytes, particularly monocytes, macrophages, and T cells, to sites of inflammation (Charo, IF and Taubman, MB (2004) Circ. Res. 95, 858-866). In addition to its role, convincing evidence, for example in vascular disease, indicates the role of MCP-1 in Alzheimer's disease (AD) (Xia, MQ and Hyman, BT (1999) J Neurovirol. 5, 32-41 ). The presence of MCP-1 in senile plaques and in reactive microglia, CNS resident macrophages were observed in the brain of patients suffering from AD (Ishizuka, K. et al. (1997) Psychiatry Clin. Neurosci. 51, 135-138). Stimulation of monocytes and microglia with amyloid-β protein (Aβ) induces chemokine secretion in vitro (Meda, L. et al. (1996) J Immunol 157, 1213-1218; Szczepanik, AM et al. (2001) J Neuroimmunol. 113, 49-62), Intraventricular injection of Aβ _(1-42) into mouse hippocampus significantly increases MCP-1 in vivo. Moreover, Aβ deposition attracts and activates microglial cells, causing them to produce inflammatory mediators such as MCP-1, which in turn induce further chemotaxis, activation, and tissue damage. To cause feedback. At the site of Aβ deposition, activated microglia also phagocytose Aβ peptides, causing amplification of activation (Rogers, J. and Lue, LF (2001) Neurochem. Int. 39, 333-340).

  Examination of chemokine expression in a 3xTg mouse model for AD revealed that neuronal inflammation preceded plaque formation and that MCP-1 was upregulated 11-fold. Furthermore, upregulation of MCP-1 appears to correlate with the occurrence of initial intracellular Aβ deposition (Janelsins, M.C. et al. (2005) J Neuroinflammation. 2, 23). A cross between a MCP-1 overexpressing mouse model and a Tg2575 mouse model for AD shows an increase in microglia accumulation around Aβ deposits, which is scattered compared to a single transgenic Tg2576 littermate Accompanied by an increase in the amount of sexual plaque (Yamamoto, M. et al. (2005) Am. J Pathol. 166, 1475-1485).

  MCP-1 levels are increased in the CSF of AD patients and patients with mild cognitive impairment (MCI) (Galimberti, D. et al. (2006) Arch. Neurol. 63, 538-543). Furthermore, MCP-1 shows increased levels in the serum of MCI and early AD patients (Clerici, F. et al. (2006) Neurobiol. Aging 27, 1763-1768).

(Summary of the Invention)
The present invention relates to inhibitors of glutaminyl peptide cyclotransferase and its use for the treatment and / or prevention of diseases or disorders selected from the group consisting of inflammatory diseases selected from:
neurodegenerative diseases such as mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down syndrome, familial British dementia, familial Danish dementia, multiple sclerosis,
b. Chronic and acute inflammation such as rheumatoid arthritis, atherosclerosis, restenosis, pancreatitis,
c. Fibrosis, such as pulmonary fibrosis, liver fibrosis, kidney fibrosis,
d. Cancer, eg cancer / angioendothelioma growth, gastric carcinoma,
e. metabolic diseases such as hypertension,
f. and other inflammatory diseases such as neuropathic pain, graft rejection / graft failure / graft vascular disorder, HIV infection / AIDS, pregnancy toxemia, tuberous sclerosis.

Shown is incubation of MCP-1 _(1-76) with N-terminal glutaminyl (A) or pyroglutamyl (5-oxo-L-prolyl) residue (B) with recombinant human DP4 for 24 hours. For cyclization of N-terminal glutamine to pyroglutamic acid, MCP-1 was incubated with recombinant human QC 3 hours before the start of the assay. DP4 cleavage products were analyzed after 0 min, 15 min, 30 min, 1 h, 4 h, and 24 h using Maldi-TOF mass spectrometry. _Shown is 24-hour incubation of human synovial fibroblast MMP-1 with MCP-1 _(1-76) having an N-terminal glutaminyl (A) or pyroglutamyl (5-oxo-L-prolyl) residue. For cyclization of N-terminal glutamine to pyroglutamic acid, MCP-1 was incubated with recombinant human QC 3 hours before the start of the assay. MMP-1 cleavage products were analyzed after 0 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 24 hours using Maldi-TOF mass spectrometry. 24-hour human synovial fibroblast MMP-1 of MCP-1 _(1-76) with N-terminal glutaminyl (A) or pyroglutamyl (5-oxo-L-prolyl) residues, and recombinant human DP4 Incubation with is shown. For cyclization to N-terminal glutamine pyroglutamate, MCP-1 was incubated with recombinant human QC 3 hours before the start of the assay. The resulting MMP-1 cleavage products were analyzed after 0 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 24 hours using Maldi-TOF mass spectrometry. 2 shows the isolation of human MCP-1 from human neuroblastoma cell line SH-SY5Y. (M: DNA standard in bp; 1: full length human MCP-1 isolated from SH-SY5Y). The nucleotide (A) and amino acid (B) alignment of human MCP-1 isolated from SH-SY5Y (upper lane) and human MCP-1 GenBank accession M24545 (lower lane) is shown. Single nucleotide polymorphisms are shown in bold font. C: human MCP-1 _(1-76) (WT) in the supernatant of transfected HEK293 cells compared to the vector-transfected control (pcDNA) and lacking the N-terminal pGlu residue (Δ = Q1) Indicates the concentration of mutant human MCP-1. (Ns: no significant difference, student t-test; n = 6). D: Migration of THP-1 monocytes toward the produced supernatant of HEK293 cells transfected at dilutions 1: 1, 1: 3, 1:10, and 1:30. (*, P <0.05; **, P <0.01; ***, P <0.001; Student t-test, n = 3). A: Human MCP-1 _(1-76) (WT) in the supernatant of transfected HEK293 cells compared to the vector-transfected control (pcDNA) and suddenly lacking two N-terminal amino acids (ΔQ1P2) The concentration of mutant human MCP-1 is shown. (**, P <0.01; Student t test; n = 6). B: Migration of THP-1 monocytes towards the produced supernatant of HEK293 cells transfected at dilutions 1: 1, 1: 3, 1:10, and 1:30. (*, P <0.05; **, P <0.01; ***, P <0.001; Student t test, n = 3). A: absence of 10 μM 1- (3- (H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea compared to the vector-transfected control (pcDNA), And the concentration of human MCP-1 _(1-76) (WT) in the supernatant of transfected HEK293 cells in the presence. (Ns: no significant difference; Student t test; n = 6). B: 10 μM 1- (3- (1H-imidazol-1-yl) propyl) hydrochloride-3- (3,4-dimethoxyphenyl) at dilutions of 1: 1, 1: 3, 1:10, and 1:30 ) Migration of THP-1 monocytes towards the produced supernatant of transfected HEK293 cells in the absence and presence of thiourea. (**, P <0.01; Student t test, n = 3). Untreated ApoE3 Leiden mice (black bars) and mice treated with 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride (white bars) Figure 2 shows quantification of vascular remodeling of a cuffed vessel wall segment. Mice were sacrificed 14 days after cuff placement. The circumference of the blood vessel (A), that is, the total area within the outer diameter of the blood vessel segment, and the remaining lumen (B) are expressed in μm ² . Untreated ApoE3 Leiden mice (black bars) or mice treated with 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride (white bars) Figure 2 shows quantification of vascular remodeling of a cuffed vessel wall segment. Mice were sacrificed 14 days after cuff placement. Lumen stenosis A is expressed in%, and the neointimal B region is expressed in μm ² . (*, P <0.05, Student t test). Untreated ApoE3 Leiden mice (black bars) or mice treated with 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride (white bars) Figure 2 shows quantification of vascular remodeling of a cuffed vessel wall segment. Mice were sacrificed 14 days after cuff placement. The area of media A is in μm ² and the intima / media ratio B is shown (*, P <0.05, Student t test). Adhesion per section in the absence (black bar) or presence (white bar) of 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride treatment Cells and infiltrating cells are shown. The total number of adherent cells per section was counted in the section of the cuffed femoral artery collected 2 days after cuff placement. Within the total population of adherent cells, monocyte / macrophage specific staining was used to identify adherent and infiltrating monocytes. (*, P <0.05, Student t test). Early time points (2 days) in untreated mice (control) and mice treated with 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride ), And an example of MCP-1 staining by immunohistochemistry of the lesion at the late time point (14 days). Absence (black bar) and presence (white bar) of the 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride treatment in the middle film, and Quantification of MCP-1 staining in cross sections of mice sacrificed after 2 days (early time point) A or 14 days (late time point) B within the neointima. (*, P <0.05; Student t test). Absence (black bar) and presence (white bar) of the 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride treatment in the middle film, and Shown is the relative amount of MCP-1 staining in sections of mice sacrificed after 2 days (early time point) (A) or 14 days (late time point) (B) within the neointima (%). (*, P <0.05; Student t test). FIG. 5 shows quantification of the promotion of atherosclerosis in the vessel wall based on quantification of monocytes / macrophages staining using the marker AIA31240. The late time point of treatment in the absence (black bar) and presence (white bar) of 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride 14) shows a cross section of a mouse sacrificed. Foam cell accumulation is illustrated as (A)% foam cell positive region / cross section and (B) μm ² as foam cell positive region / cross section. _Illustrates cleavage of human MCP-1 _(1-76) with N-terminal glutaminyl (A) or pyroglutamyl (5-oxo-L-prolyl) residue (B) by 24-hour recombinant human aminopeptidase P . Pyroglutamate formation at the N-terminus was achieved by incubating MCP-1 with recombinant human QC 3 hours prior to the assay. DP4 cleavage products were analyzed after 0 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 24 h using Maldi-TOF mass spectrometry. 4 illustrates cleavage of human MCP-1 _(1-76) with N-terminal glutaminyl (A), or pyroglutamyl (5-oxo-L-prolyl) residue (B) by 4 hours of recombinant human DP4. Pyroglutamate formation at the N-terminus was achieved by incubating MCP-1 with recombinant human QC 3 hours prior to the assay. In addition, incubation of Gln ¹ -MCP-1 with recombinant human QC was performed with 1- (3- (1H-imidazol-1-yl) propyl hydrochloride) -3- (3, a 10 μM QC-specific inhibitor. , 4-dimethoxyphenyl) thiourea. DP4 cleavage products were analyzed after 0 min, 15 min, 30 min, 1 h, 2 h, and 4 h using Maldi-TOF mass spectrometry. Degradation of human MCP-1 _(1-76) with N-terminal glutaminyl residue (A) or pyroglutamyl (5-oxo-L-prolyl) residue (B) in human serum at 7 and 24 hours, respectively Show. For cyclization of the N-terminal glutamine residue to pyroglutamic acid, MCP-1 was incubated with recombinant human QC 3 hours before the start of the assay. In addition, Gln ¹ -MCP-1 was incubated in human serum for 24 hours in the presence of the 9.6 μM DP4 inhibitor isoleucyl-thiazolidide (P32 / 98) (C). Cleavage products were analyzed using Maldi-TOF mass spectrometry for Gln ¹ -MCP-1 after 0 min, 10 min, 30 min, 1 h, 2 h, 3 h, 5 h, and 7 h. For pGlu1-MCP-1, after 0 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 7 hours, and 24 hours, and for Gln ¹ -MCP-1 in combination with isoleucyl-thiazolidide, Analysis was performed after 0 minute, 1 hour, 2 hours, 3 hours, 5 hours, 7 hours, and 24 hours. FIG. 4 shows degradation of human MCP-2 _(1-76) with N-terminal glutaminyl (A) or pyroglutamyl (5-oxo-L-prolyl) residue (B) by 24 hours of recombinant human DP4. For cyclization of N-terminal glutamine to pyroglutamic acid, MCP-2 was incubated with recombinant human QC 3 hours before the start of the assay. DP4 cleavage products were analyzed after 0 min, 15 min, 30 min, 1 h, 2 h, 4 h and 24 h using Maldi-TOF mass spectrometry. _{Figure 6} shows degradation of human MCP-3 _(1-76) with N-terminal glutaminyl (A) or pyroglutamyl (5-oxo-L-prolyl) residue (B) by 24 hours of recombinant human DP4. For cyclization of N-terminal glutamine to pyroglutamic acid, MCP-3 was incubated with recombinant human QC 3 hours before the start of the assay. DP4 cleavage products were analyzed after 0 min, 15 min, 30 min, 1 h, 2 h, 4 h and 24 h using Maldi-TOF mass spectrometry. 4 illustrates cleavage of human MCP-4 _(1-75) with N-terminal glutaminyl (A) or pyroglutamyl (5-oxo-L-prolyl) residue (B) by 4 hours of recombinant human DP4. For cyclization of N-terminal glutamine to pyroglutamic acid, MCP-4 was incubated with recombinant human QC 3 hours prior to the start of the assay. DP4 cleavage products were analyzed after 0 min, 15 min, 30 min, 1 h, 2 h, and 4 h using Maldi-TOF mass spectrometry. N-terminal glutamine (Gln ¹ -MCP-1), proglutamate (pGlu ¹ -MCP-1) (5-oxo-L-proline), proline 2 (Pro ² -MCP-1, aminopeptidase P cleavage product) Of human N-terminal MCP-1 mutants beginning with aspartic acid 3 (Asp ³ -MCP-1, DP4 cleavage product) and beginning with isoleucine 5 (Ile ⁵ -MCP-1, MMP-1 cleavage product) 2 shows chemotactic ability for human THP-1 monocytes. Chemotaxis of human MCP-1 incubated with human recombinant DP4 in the presence (Gln ¹ -MCP-1 + QC + DP4) and absence (Gln ¹ -MCP-1 + DP4) in the presence of QC-mediated pGlu formation Shows analysis of capabilities. In addition, N-terminal pGlu-residue of 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea (QCI) (10 μM), a QC inhibitor The effect on group formation and subsequent DP4 cleavage (Gln ¹ -MCP-1 + QC + QCI + DP4) is shown. The chemotactic ability of human MCP-1 (A), MCP-2 (B), MCP-3 (C), and MCP-4 (D) in the absence or presence of N-terminal pyroglutamyl residues is shown. The chemotaxis potential of full-length human MCP-1 (A), MCP-3 (B), MCP-2 (C), and MCP-4 (D) starting with N-terminal glutamine is compared with their respective DP4 cleavage products. Shown in comparison. TNFα after application of the QC inhibitor 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea in a model of LPS-induced sepsis in rats Shows a significant decrease in levels (ANOVA, P <0.05). Figure 3 shows a dose-dependent decrease in monocyte infiltration into the peritoneum in a model of peritonitis induced in mice by thioglycolate caused by a QC inhibitor. Thioglycolate and QCI (1- (3- (1H-imidazol-1-yl) propyl hydrochloride) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride) at 25 mg / kg, 50 mg / kg, and 100 mg / Three different concentrations of kg were injected. Peritoneal infiltrating cells were classified using FACS analysis 4 hours after inducing peritonitis. (*, P <0.05, Student t test). Thioglycolate exposure in combination with QCI (1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea), a QC-specific inhibitor The decrease in Moma2-positive cells in the peritoneal lavage fluid of mice is shown compared to animals not receiving QCI (*, P <0.05, Student t test).

(Detailed description of the invention)
In particular, the present invention belongs to the following items:
1. a QC inhibitor for the treatment and / or prevention of an inflammatory disease or condition selected from:
neurodegenerative diseases including mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down syndrome, familial British dementia, familial Danish dementia, and multiple sclerosis,
b. chronic and acute inflammation, including rheumatoid arthritis, atherosclerosis, restenosis, and pancreatitis,
c. Fibrosis including pulmonary fibrosis, liver fibrosis, and kidney fibrosis
d. Cancers including cancer / angioendothelioma growth and gastric carcinoma,
e. Metabolic diseases including hypertension,
f. and other inflammatory diseases including neuropathic pain, graft rejection / graft failure / graft vasculopathy, HIV infection / AIDS, pregnancy toxemia, tuberous sclerosis.

  2. The QC inhibitor according to item 1, wherein the neurodegenerative disease is selected from mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down syndrome, familial British dementia, familial Danish dementia, and multiple sclerosis .

  3. The QC inhibitor according to item 1 or 2, wherein the disease is mild cognitive impairment.

  4. QC inhibitors are nootropics, neuroprotectives, antiparkinsonian drugs, amyloid protein deposition inhibitors, beta amyloid synthesis inhibitors, antidepressants, anxiolytics, antipsychotics, and anti-multiple sclerosis 4. The QC inhibitor according to any of items 1 to 3, wherein the QC inhibitor is administered in combination with an additional agent selected from the group consisting of drugs.

  5. The QC according to item 1, wherein the disease is chronic or acute inflammation selected from rheumatoid arthritis, atherosclerosis, restenosis and pancreatitis.

  6. The QC inhibitor according to item 1 or 5, wherein the disease is selected from restenosis and pancreatitis.

  7. QC inhibitor is an angiotensin converting enzyme (ACE) inhibitor; angiotensin II receptor blocker; diuretic; calcium channel blocker (CCB); beta blocker; platelet aggregation inhibitor; cholesterol absorption modulator; Co-A reductase inhibitors; compounds that increase high density lipoprotein (HDL); renin inhibitors; IL-6 inhibitors; anti-inflammatory corticosteroids; antiproliferative drugs; nitric oxide donors; Inhibitor; growth factor or cytokine signaling inhibitor; QC inhibition according to item 1, 5, or 6 administered in combination with an additional agent selected from the group consisting of an MCP-1 antagonist and a tyrosine kinase inhibitor Agent.

8. Use of a QC inhibitor to treat and / or prevent an inflammatory disease or condition selected from:
neurodegenerative diseases including mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down syndrome, familial British dementia, familial Danish dementia, multiple sclerosis,
b. chronic and acute inflammation, including rheumatoid arthritis, atherosclerosis, restenosis, pancreatitis,
c. Fibrosis including pulmonary fibrosis, liver fibrosis, kidney fibrosis
d. Cancer / cancer including hemangioendothelioma growth, gastric carcinoma,
e. metabolic diseases including hypertension,
f. and other inflammatory diseases including neuropathic pain, graft rejection / graft failure / graft vascular disorder, HIV infection / AIDS, pregnancy toxemia, tuberous sclerosis.

  9. Item 8 is a neurodegenerative disease selected from mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down syndrome, familial British dementia, familial Danish dementia, multiple sclerosis Use of.

  10. Use according to item 8 or 9, wherein the disease is mild cognitive impairment (MCI).

  11. QC inhibitors are nootropics, neuroprotectives, antiparkinsonian drugs, amyloid protein deposition inhibitors, beta amyloid synthesis inhibitors, antidepressants, anxiolytics, antipsychotics, and anti-multiple sclerosis 11. Use according to any of items 8-10, administered in combination with a further agent selected from the group consisting of drugs.

  12. The use according to item 8, wherein the disease is chronic or acute inflammation selected from rheumatoid arthritis, atherosclerosis, restenosis and pancreatitis.

  13. The use according to item 8 or 12, wherein the disease is selected from restenosis and pancreatitis.

  14. QC inhibitors are inhibitors of angiotensin converting enzyme (ACE); angiotensin II receptor blockers; diuretics; calcium channel blockers (CCB); beta blockers; platelet aggregation inhibitors; cholesterol absorption modulators; -A reductase inhibitors; compounds that increase high density lipoprotein (HDL); renin inhibitors; IL-6 inhibitors; anti-inflammatory corticosteroids; antiproliferative drugs; nitric oxide donors; 14. Use according to item 8, 12, or 13, wherein said agent is administered in combination with a further agent selected from the group consisting of an agent; a growth factor or cytokine signaling inhibitor; an MCP-1 antagonist, and a tyrosine kinase inhibitor.

15. Use of a QC inhibitor for the manufacture of a medicament for treating and / or preventing an inflammatory disease or condition selected from:
neurodegenerative diseases including mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down syndrome, familial British dementia, familial Danish dementia, and multiple sclerosis,
b. chronic and acute inflammation, including rheumatoid arthritis, atherosclerosis, restenosis, pancreatitis,
c. Fibrosis including pulmonary fibrosis, liver fibrosis, kidney fibrosis
d. Cancer / cancer including hemangioendothelioma growth, gastric carcinoma,
e. metabolic diseases including hypertension,
f. and other inflammatory diseases including neuropathic pain, graft rejection / graft failure / graft vascular disorder, HIV infection / AIDS, pregnancy toxemia, tuberous sclerosis.

  16. The disease according to item 15, wherein the disease is a neurodegenerative disease selected from mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down syndrome, familial British dementia, familial Danish dementia, multiple sclerosis use.

  17. Use according to item 15 or 16, wherein the disease is mild cognitive impairment (MCI).

  18. QC inhibitors are nootropics, neuroprotective drugs, antiparkinsonian drugs, amyloid protein deposition inhibitors, beta amyloid synthesis inhibitors, antidepressants, anxiolytics, antipsychotics, and anti-multiple sclerosis 18. Use according to any of items 15-17, administered in combination with a further agent selected from the group consisting of drugs.

  19. The use according to item 15, wherein the disease is chronic or acute inflammation selected from rheumatoid arthritis, atherosclerosis, restenosis, and pancreatitis.

  20. Use according to item 15 or 19, wherein the disease is selected from restenosis and pancreatitis.

  21. QC inhibitors are inhibitors of angiotensin converting enzyme (ACE); angiotensin II receptor blockers; diuretics; calcium channel blockers (CCB); beta blockers; platelet aggregation inhibitors; cholesterol absorption modulators; Co-A reductase inhibitors; compounds that increase high density lipoprotein (HDL); renin inhibitors; IL-6 inhibitors; anti-inflammatory corticosteroids; antiproliferative drugs; nitric oxide donors; 21. Any of items 15, 19, or 20 administered in combination with an additional agent selected from the group consisting of an inhibitor; a growth factor or cytokine signaling inhibitor; an MCP-1 antagonist, and a tyrosine kinase inhibitor Use of.

22. a. Neurodegenerative diseases including mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down syndrome, familial British dementia, familial Danish dementia, and multiple sclerosis,
b. chronic and acute inflammation, including rheumatoid arthritis, atherosclerosis, restenosis, pancreatitis,
c. Fibrosis including pulmonary fibrosis, liver fibrosis, kidney fibrosis
d. Cancer / cancer including hemangioendothelioma growth, gastric carcinoma,
e. metabolic diseases including hypertension,
f. and inflammatory selected from other inflammatory diseases including neuropathic pain, graft rejection / graft failure / graft vascular disorder, HIV infection / AIDS, pregnancy toxemia, tuberous sclerosis A method of treating and / or preventing a disease or condition, wherein an effective amount of a QC inhibitor is administered to a subject in need thereof.

  23. The item according to item 22, wherein the disease is a neurodegenerative disease selected from mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down syndrome, familial British dementia, familial Danish dementia, multiple sclerosis Methods of treatment and / or prevention.

  24. The method of treatment and / or prevention according to item 23 or 24, wherein the disease is mild cognitive impairment (MCI).

  25. QC inhibitors are nootropics, neuroprotectives, antiparkinsonian drugs, amyloid protein deposition inhibitors, beta amyloid synthesis inhibitors, antidepressants, anxiolytics, antipsychotics, and anti-multiple sclerosis 26. The method of treatment and / or prevention according to any of items 23 to 25, wherein the method is administered in combination with a further agent selected from the group consisting of drugs.

  26. The method for treatment and / or prevention according to item 23, wherein the disease is chronic or acute inflammation selected from rheumatoid arthritis, atherosclerosis, restenosis, and pancreatitis.

  27. The method of treatment and / or prevention according to item 23 or 26, wherein the disease is selected from restenosis and pancreatitis.

  28. QC inhibitors are inhibitors of angiotensin converting enzyme (ACE); angiotensin II receptor blockers; diuretics; calcium channel blockers (CCB); beta blockers; platelet aggregation inhibitors; cholesterol absorption modulators; Co-A reductase inhibitors; compounds that increase high density lipoprotein (HDL); renin inhibitors; IL-6 inhibitors; anti-inflammatory corticosteroids; antiproliferative drugs; nitric oxide donors; 28. The treatment of item 23, 26, or 27, administered in combination with an additional agent selected from the group consisting of: an inhibitor; a growth factor or cytokine signaling inhibitor; an MCP-1 antagonist, and a tyrosine kinase inhibitor; And / or methods of prevention.

  29. Use according to any of items 7 to 21, wherein the disease and / or condition afflicts a human.

  30. The method of any of items 22-28, wherein the disease and / or condition afflicts a human.

  31. Any one of the preceding items, wherein the QC inhibitor is an inhibitor selected from Formulas 1, 1 *, 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, and 1i. Use or method.

  32. The use or method according to any one of items 1-31, wherein the QC inhibitor is an inhibitor selected from Examples 1-141.

  33. Any one of Items 1-32, wherein the QC inhibitor is 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxy-phenyl) thiourea hydrochloride The use or method described in 1.

  34. Diagnostic assays involving QC inhibitors.

  35. The diagnostic assay of item 34, wherein the QC inhibitor is an inhibitor selected from formulas 1, 1 *, 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, and 1i.

  36. The diagnostic assay of item 34 or 35, wherein the QC inhibitor is an inhibitor selected from Examples 1-141.

  37. The item according to any one of items 34 to 36, wherein the QC inhibitor is 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxy-phenyl) thiourea hydrochloride. Diagnostic assay.

38. A method for diagnosing any one of the diseases and / or conditions defined in item 1;
-Collecting a sample from a subject suspected of suffering from said disease and / or condition;
-Contacting the sample with a QC inhibitor; and
-Determining whether the subject is afflicted by the disease and / or condition;
Said method.

  39. The method of item 38, wherein the subject is a human.

  40. The method of item 38 or 39, wherein the QC inhibitor is an inhibitor selected from formulas 1, 1 *, 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, and 1i.

  41. The method of any of items 38-40, wherein the QC inhibitor is an inhibitor selected from Examples 1-141.

  42. Any of paragraphs 38-41, wherein the QC inhibitor is 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxy-phenyl) thiourea hydrochloride. Method.

  43. The method according to any of items 38 to 42, wherein the sample is a blood sample, serum sample, cerebrospinal solution sample, or urine sample.

  44. A diagnostic kit for carrying out the method of items 38 to 42, comprising the diagnostic assay according to any of items 34 or 37 as the detection means, and the determination means.

  45. A pharmaceutical composition comprising the QC inhibitor according to any one of items 1 to 7 or 31 to 33.

  In particularly preferred embodiments, the present invention treats chronic or acute inflammation selected from rheumatoid arthritis, atherosclerosis, restenosis, and pancreatitis, particularly restenosis and pancreatitis, most preferably restenosis. Relates to the use of QC inhibitors in methods.

  The effects of QC inhibitors for treating chronic or acute inflammation selected from rheumatoid arthritis, atherosclerosis, restenosis, and pancreatitis are described in Examples 3, 7, and 8 of the present invention. Can be tested using in vivo assays.

  More preferably according to the present invention is the use of a QC inhibitor in a method of treating mild cognitive impairment (MCI).

Therefore, the present invention more preferably belongs to the following items:
1. a QC inhibitor for the treatment and / or prevention of an inflammatory disease or condition selected from mild cognitive impairment (MCI), restenosis, and pancreatitis.

  2. Use of a QC inhibitor for the treatment and / or prevention of an inflammatory disease or condition selected from mild cognitive impairment (MCI), restenosis, and pancreatitis.

  3. Use of a QC inhibitor for the manufacture of a medicament for treating and / or preventing an inflammatory disease or condition selected from mild cognitive impairment (MCI), restenosis, and pancreatitis.

  4. The QC inhibitor or use according to any one of items 1 to 3, wherein the disease is mild cognitive impairment (MCI).

  5. QC inhibitors are nootropics, neuroprotective agents, antiparkinsonian drugs, amyloid protein deposition inhibitors, beta amyloid synthesis inhibitors, antidepressants, anxiolytics, antipsychotics, and anti-multiple sclerosis 5. A QC inhibitor according to any of items 1 to 4, or use, wherein the QC inhibitor is administered in combination with a further agent selected from the group consisting of drugs.

  6. The QC inhibitor or use according to any of items 1 to 3, wherein the disease is selected from restenosis and pancreatitis.

  7. The QC inhibitor or use according to any of items 1 to 3, or 6, wherein the disease is restenosis.

  8. QC inhibitors are inhibitors of angiotensin converting enzyme (ACE); angiotensin II receptor blockers; diuretics; calcium channel blockers (CCB); beta blockers; platelet aggregation inhibitors; cholesterol absorption modulators; Co-A reductase inhibitors; compounds that increase high density lipoprotein (HDL); renin inhibitors; IL-6 inhibitors; anti-inflammatory corticosteroids; antiproliferative drugs; nitric oxide donors; Any of items 1-3, 6, or 7, administered in combination with an additional agent selected from the group consisting of an inhibitor; a growth factor or cytokine signaling inhibitor; an MCP-1 antagonist, and a tyrosine kinase inhibitor Or a QC inhibitor or use.

  9. A method for the treatment and / or prevention of an inflammatory disease or condition selected from mild cognitive impairment (MCI), restenosis, and pancreatitis, in which an effective amount of a QC inhibitor is in need thereof Said method of administration.

  10. The method of treatment and / or prevention according to item 9, wherein the disease is mild cognitive impairment (MCI).

  11. QC inhibitors are nootropics, neuroprotectives, antiparkinsonian drugs, amyloid protein deposition inhibitors, beta amyloid synthesis inhibitors, antidepressants, anxiolytics, antipsychotics, and anti-multiple sclerosis 11. The method of treatment and / or prevention according to item 9 or 10, wherein the method is administered in combination with an additional drug selected from the group consisting of drugs.

  12. The method of treatment and / or prevention according to item 9, wherein the disease is chronic or acute inflammation selected from rheumatoid arthritis, atherosclerosis, restenosis and pancreatitis.

  13. The method of treatment and / or prevention according to item 9 or 12, wherein the disease is selected from restenosis and pancreatitis.

  14. The method of treatment and / or prevention according to any of items 9, 12, or 13, wherein the disease is restenosis.

  15. QC inhibitor is an angiotensin converting enzyme (ACE) inhibitor; angiotensin II receptor blocker; diuretic; calcium channel blocker (CCB); beta blocker; platelet aggregation inhibitor; cholesterol absorption modulator; Co-A reductase inhibitors; compounds that increase high density lipoprotein (HDL); renin inhibitors; IL-6 inhibitors; anti-inflammatory corticosteroids; antiproliferative drugs; nitric oxide donors; 15. Inhibitor; Growth factor or cytokine signaling inhibitor; MCP-1 antagonist and any of items 9 or 12-14 administered in combination with an additional agent selected from the group consisting of a tyrosine kinase inhibitor For the treatment and / or prevention.

  16. Use according to any of items 2 to 8, wherein the disease and / or condition afflicts a human.

  17. The method of any of paragraphs 9-15, wherein the disease and / or condition afflicts a human.

  18. In any one of Items 1-17, wherein the QC inhibitor is an inhibitor selected from Formulas 1, 1 *, 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, and 1i A QC inhibitor, use, or method as described.

  19. The QC inhibitor, use, or method according to any one of items 1-18, wherein the QC inhibitor is an inhibitor selected from Examples 1-141.

  20. Any one of Items 1-19, wherein the QC inhibitor is 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxy-phenyl) thiourea hydrochloride A QC inhibitor, use, or method as described in.

  21. Diagnostic assays involving QC inhibitors.

  22. The diagnostic assay of item 21, wherein the QC inhibitor is an inhibitor selected from Formulas 1, 1 *, 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, and 1i.

  23. The diagnostic assay of item 21 or 22, wherein the QC inhibitor is an inhibitor selected from Examples 1-141.

  24. The item according to any of items 21 to 23, wherein the QC inhibitor is 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxy-phenyl) thiourea hydrochloride Diagnostic assay.

25. A method for diagnosing any one of the diseases and / or conditions defined in item 1;
-Collecting a sample from a subject suspected of suffering from said disease and / or condition;
Contacting said sample with an inhibitor of glutaminyl peptide cyclotransferase; and
-Determining whether the subject is afflicted by the disease and / or condition;
Said method.

  26. The method of item 26, wherein the subject is a human.

  27. The method according to item 26 or 27, wherein the QC inhibitor is an inhibitor selected from the formulas 1, 1 *, 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, and 1i.

  28. The method according to any of items 25 to 27, wherein the QC inhibitor is an inhibitor selected from Examples 1-141.

  29. The item according to any one of Items 25 to 28, wherein the QC inhibitor is 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxy-phenyl) thiourea hydrochloride. Method.

  30. The method of any of items 25-29, wherein the sample is a blood sample, a serum sample, a cerebrospinal solution sample, or a urine sample.

  31. A diagnostic kit for carrying out the method of items 25 to 30, comprising the diagnostic assay according to any of items 21 to 24 as a detection means, and the determination means.

  32. A pharmaceutical composition comprising the QC inhibitor according to any one of items 1, 4 to 6, or 18 to 20.

(Definition)
(Enzyme inhibitors, especially QC inhibitors)
Reversible enzyme inhibitors: include competitive inhibitors, non-competitive reversible inhibitors, slow or tihgt inhibitors, transition state analogs, and multi-substrate analogs.

Competitive inhibitors are
i) non-covalent interaction with the enzyme,
ii) competition with the substrate for the enzyme active site,
Indicates.

The main mechanism of action of reversible enzyme inhibitors and the definition of dissociation constants can be visualized as follows:

  Formation of the enzyme inhibitor [E-I] complex prevents substrate binding and therefore the reaction cannot proceed to the normal physiological product (P). Higher inhibitor concentration [I] results in higher [E-I], leaving less free enzyme to which the substrate can bind.

Noncompetitive reversible inhibitor
i) Bind to sites other than the active site (allosteric binding site)
ii) causing a conformational change in the enzyme that reduces or stops catalytic activity.

Slow binding or tight binding inhibitor
i) a competitive inhibitor in which the equilibrium between the inhibitor and the enzyme is slowly achieved;
ii) probably due to conformational changes that must occur in the enzyme or inhibitor (k _on is slow)
a) Usually a transition state analogue
b) Effective at the same concentration as the enzyme concentration (KD value of nM or less)
c) This kind of inhibitor is “almost” irreversible because the k _off value is very low.

Transition state analogs Competitive inhibitors that mimic the transition state of enzyme-catalyzed reactions. Thus, enzyme catalysis is caused by a decrease in the energy of the transition state, and thus transition state binding is selected over substrate binding.

Multi-substrate analog
For reactions involving more than one substrate, competitive inhibitors, or transition state analogs that contain structural features resembling more than one substrate can be designed.

  (Irreversible enzyme inhibitor): drive the unbound enzyme and the equilibrium between the inhibitor and the enzyme inhibitor complex (E + I <---> EI) completely to the EI side by covalent bond (~ 100 kcal / mol), causing irreversible inhibition.

Affinity-labeled agent Active site directed irreversible inhibitors (competitive irreversible inhibitors) are recognized by enzymes (reversible, specific binding), followed by covalent bond formation, and
i) structurally similar to a substrate, transition state, or product and capable of specifically interacting between the drug and the target enzyme
ii) It contains a reactive functional group (eg, a nucleophile, —COCH ₂ Br) and can form a covalent bond.

Reaction Scheme below, describes an active site directed reagent with its target enzyme, wherein, K _D is the dissociation constant, and k _Inactivation is the rate of covalent bond formation.

  Mechanism-based enzyme inactivators (also called suicide inhibitors) are active site binding agents that bind to an enzyme active site where they are transformed into a reactive form (activated) by the catalytic ability of the enzyme. Directional reagent (non-reactive). Once activated, a covalent bond is formed between the inhibitor and the enzyme.

Reaction scheme below shows the mechanism of action of the enzyme inactivating agent, based on the mechanism, K _D where is the dissociation complex, k _2, once the rate of activation of the inhibitor bound to the enzyme K ₃ is the rate of dissociation of the activated inhibitor P from the enzyme (the product can still be reactive) and k ₄ is the activated inhibitor and enzyme The rate of covalent bond formation between

Inactivation (covalent bond formation, k ₄ ) must occur before dissociation (k ₃ ), otherwise the reactive inhibitor is released into the environment. Partition ratio, k ₃ / k ₄ : The ratio of released product to inactivation should be minimized for efficient inactivation of the system and minimal unwanted side reactions.

  Large partition ratios (choose dissociation) cause non-specific reactions.

(Uncompetitive enzyme inhibitor): As a definition of uncompetitive inhibitor (inhibitor that binds only to the ES complex), the following equilibrium equation can be assumed:

  The ES complex separates the substrate with the same dissociation constant as Ks, but the ESI complex does not separate it (ie has a Ks value equal to zero). The Km of Michaelis-Menten enzyme is expected to be reduced. Increasing the substrate concentration causes an increase in ESI concentration (complex that cannot progress to the reaction product), and thus inhibition cannot be removed.

  Competitive enzyme inhibitors are preferred in the present invention.

  Competitive reversible enzyme inhibitors are most preferred.

The terms “K _i ”, or “K _I ”, and “K _D ” are binding constants that describe the binding of an inhibitor to an enzyme and subsequent release. Another measure is the “IC ₅₀ ” value, which reflects the inhibitor concentration and produces 50% enzyme activity at a given substrate concentration.

(QC)
As used herein, the term “QC” includes glutaminyl cyclase (QC) and QC-like enzymes. QC and QC-like enzymes have the same or similar enzyme activity and are further defined as QC activity. In this regard, QC-like enzymes can differ fundamentally from QC in their molecular structure.

  As used herein, the term “QC activity” refers to N-terminal glutamyl residues to pyroglutamic acid (pGlu *), or N-terminal L-homoglutaminyl, or L-β-homoglutaminyl, under the release of ammonia. Defined as intramolecular cyclization to cyclic pyro-homoglutamine derivatives. See Schemes 1 and 2 in this regard.

本明細書に使用される「EC」という用語は、EC活性として更に定義される、グルタミン酸シクラーゼ（EC）としてのQC、及びQC様酵素の副活性を含む。

As used herein, the term “EC” includes QC as glutamate cyclase (EC), which is further defined as EC activity, and side-activity of QC-like enzymes.

  The term “EC activity” as used herein is defined as intramolecular cyclization of the N-terminal glutamyl residue to pyroglutamic acid (pGlu *) by QC. See Scheme 3 in this regard.

  The terms “QC inhibitor”, “glutaminyl cyclase inhibitor” are generally known to those skilled in the art and generally inhibit glutamate cyclase (QC), or its catalytic activity of glutamyl cyclase (EC) activity, generally as described above. Means an enzyme inhibitor as defined.

(QC inhibition ability)
In view of the correlation with QC inhibition, in preferred embodiments, in the present methods and medical applications, it is 10 μM or less, more preferably 1 μM or less, even more preferably 0.1 μM or less, or 0.01 μM or less, or most preferably Uses drugs with a Ki for QC inhibition of 0.001 μM or less. Indeed, inhibitors with Ki values below the micromolar, preferably nanomolar, and even more preferably picomolar range are envisioned. Thus, for convenience, active agents are described herein as “QC inhibitors”, but such nomenclature is not intended to limit the subject matter of the invention in any way. Will be understood.

(Molecular weight of QC inhibitor)
Generally, the QC inhibitor for this method or medical use is a molecular weight of, for example, 1000 g / mole or less, 500 g / mole or less, preferably 400 g / mole or less, and more preferably 350 g / mole or less, and even 300 g / mole or less. A small molecule with

  As used herein, the term “subject” is intended for treatment, observation or experimentation and / or an animal suspected of being afflicted with the disease and / or condition defined in the item, preferably Mammals, most preferably humans.

  As used herein, the term “therapeutically effective amount” refers to the tissue system sought by a researcher, veterinarian, medical doctor, or other clinician, including the reduction of the disease being treated or the symptoms of the disorder, The amount of active compound or pharmaceutical agent that elicits a biological or medical response in an animal or human.

  The term “pharmaceutically acceptable” as used herein includes human and veterinary uses: for example, the term “pharmaceutically acceptable” includes veterinarily acceptable compounds. Or a compound acceptable in human medicine and health care.

Pharmaceutically acceptable salts:
In view of the close relationship between the free compound and the salt or solvate form of the compound, whenever a compound or inhibitor is referred to in this context, the corresponding salt or solvent, respectively. Japanese products are also intended, provided that such is possible or appropriate under the circumstances.

  Salts and solvates of the inhibitors of the present invention, and physiologically functional derivatives thereof suitable for use in medicine, are those in which the counterion or accompanying solvent is pharmaceutically acceptable. However, salts and solvates with pharmaceutically unacceptable counterions or associated solvents are also used as intermediates in the preparation of, for example, other compounds and their pharmaceutically acceptable salts and solvates. Therefore, it is within the scope of the present invention.

  Suitable salts according to the present invention include those formed with both organic and inorganic acids or bases. Pharmaceutically acceptable acid addition salts include those formed from: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, trifluoroacetic acid, Triphenylacetic acid, sulfamic acid, sulphanilic, succinic acid, oxalic acid, fumaric acid, maleic acid, malic acid, mandelic acid, glutamic acid, aspartic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, Aryl sulfonic acids (eg, p-toluene sulfonic acid, benzene sulfonic acid, naphthalene sulfonic acid, or naphthalene disulfonic acid), salicylic acid, glutaric acid, gluconic acid, tricarballylic acid, cinnamic acid, substituted silicic acid Cinnamic acids (eg, phenyl, methyl, meth- ol including 4-methyl and 4-methoxycinnamic acid Xy- or halo-substituted cinnamic acid), ascorbic acid, oleic acid, naphthoic acid, hydroxynaphthoic acid (eg 1- or 3-hydroxy-2-naphthoic acid), naphthalene acrylic acid (eg naphthalene-2- Acrylic acid), benzoic acid, 4-methoxybenzoic acid, 2- or 4-hydroxybenzoic acid, 4-chlorobenzoic acid, 4-phenylbenzoic acid, benzeneacrylic acid (eg 1,4-benzenediacrylic acid), Isethionic acid, perchloric acid, propionic acid, glycolic acid, hydroxyethanesulfonic acid, pamoic acid, cyclohexanesulfamic acid, salicylic acid, saccharic acid, and trifluoroacetic acid. Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts such as those of sodium and potassium, alkaline earth metal salts such as those of calcium and magnesium, and dicyclohexylamine and N-methyl-D-glutamine And salts with organic bases such as

All pharmaceutically acceptable acid addition salt forms of the inhibitors of the present invention are intended to be encompassed by the scope of the present invention.
Examples of solvates include hydrates.

Polymorph crystal form:
Furthermore, some of the crystalline forms of the inhibitor may exist as polymorphs and are therefore intended to be included in the present invention. In addition, some of the compounds will form solvates with water (ie, hydrates), or common organic solvents, and such solvates are also within the scope of the present invention. It is intended to be included. Inhibitors include their salts and other solvents that can also be obtained in the form of their hydrates or used for their crystallization.

Prodrug:
The present invention further includes within its scope prodrugs of the inhibitors of the present invention. In general, such prodrugs will be functional derivatives of inhibitors that can be readily converted in vivo to the desired therapeutically active inhibitor. Thus, in these cases, in the methods of treatment of the present invention, the term “administering” is described by one or more prodrug versions of the inhibitors listed in the item, but administered to the subject. It would include the treatment of various disorders that later convert in vivo to the above identified inhibitors. For example, conventional procedures for the selection and manufacture of suitable prodrug derivatives are described, for example, in “Design of Prodrugs”, H. Bundgaard, Elsevier, 1985, and patent application DE 198 28. 113, DE 198 28 114, WO99 / 67228, and WO99 / 67279, which are fully incorporated herein by reference.

Protecting group:
During any of the methods for the preparation of the inhibitors of the present invention, it may be necessary and / or desirable to protect groups that are sensitive or reactive to any of the molecules concerned. unknown. Protective Groups in Organic Chemistry, edited by JFW, McOmie, Plenum Press, 1973; and TW Greene, and PGM Wuts, Protective Groups in Organic Synthesis, John Wiley & It may be achieved by conventional protecting groups, such as those described in Sons, 1991, and is fully incorporated herein by reference. The protecting group may be removed at a convenient subsequent stage using methods known in the art.

  As used herein, the term “composition” refers to a therapeutically effective amount of a product comprising a compound listed in an item, as well as any product resulting from a combination of compounds listed directly or indirectly. It is intended to include.

Carriers and additives for herbal preparations:
For liquid oral formulations such as suspensions, elixirs, and solutions, suitable carriers and additives conveniently include water, glycols, oils, alcohols, seasoning agents, preservatives, colorants, and the like. For solid oral formulations such as powders, capsules, gelcaps, and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrations Including agents.

  Carriers that can be added to the mixture include, but are not limited to, suitable binders, suspending agents, lubricants, flavoring agents, sweeteners, preservatives, coatings, disintegrants, pigments, and coloring agents. Contains inert pharmaceutical excipients.

  Soluble polymers as targetable drug carriers include polyvinylpyrrolidone, pyran copolymers, polyhydroxypropyl methacrylamide phenol, polyhydroxyethyl aspartamide phenol, or polyethylene oxide substituted with palmitoyl residue (s) Polylysine can be included. In addition, the inhibitors of the present invention include biodegradable polymer types useful for achieving sustained release / sustained drug release, such as polylactic acid, poly-epsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, It may be bonded to a block copolymer of polyacetal, polydihydropyran, polycyanoacrylic acid, and a crosslinked or amphiphilic hydrogel.

  Suitable binders include natural sugars such as starch, gelatin, glucose, or β-lactose, corn sweeteners, natural products such as acacia, tragacantha, or sodium oleate, and synthetic gums, sodium stearate, stearic acid Including but not limited to magnesium, sodium benzoate, sodium acetate, sodium chloride and the like.

  Disintegrants include, but are not limited to starch, methylcellulose, agar, bentonite, xanthan gum and the like.

(Example of QC inhibitor)
Suitable QC inhibitors for use and methods according to the present invention are disclosed in WO2005 / 075436, which is incorporated herein in its entirety with respect to the structure, synthesis, and method of use of QC inhibitors. .

式中、
Aは：
アルキル鎖、アルケニル鎖、若しくはアルキニル鎖か；
又はAは：

から選択される基であり：
式中：
R⁶、R⁷、R⁸、R⁹、及びR¹⁰は、独立してH、又はアルキル鎖、アルケニル鎖、アルキニル鎖、シクロアルキル、炭素環、アリール、ヘテロアリール、若しくは複素環であり；
n、及びn¹は、独立して1〜5であり；
mは、1〜5であり；
oは、0〜4であり；
かつ、Bは：

から選択される基であり、
式中：
D、及びEは、独立してアルキル鎖、アルケニル鎖、アルキニル鎖、シクロアルキル、炭素環、アリール、-アルキルアリール、ヘテロアリール、-アルキルヘテロアリール、アシル、又は複素環を表す。 The present invention provides novel inhibitors of QC (EC) of formula 1,

Where
A:
An alkyl chain, an alkenyl chain, or an alkynyl chain;
Or A:

Is a group selected from:
In the formula:
R ⁶ , R ⁷ , R ⁸ , R ⁹ , and R ¹⁰ are independently H, or an alkyl chain, alkenyl chain, alkynyl chain, cycloalkyl, carbocycle, aryl, heteroaryl, or heterocycle;
n and n ¹ are independently 1 to 5;
m is 1-5;
o is 0-4;
And B is:

A group selected from
In the formula:
D and E independently represent an alkyl chain, alkenyl chain, alkynyl chain, cycloalkyl, carbocycle, aryl, -alkylaryl, heteroaryl, -alkylheteroaryl, acyl, or heterocycle.

X represents CR ²⁰ R ²¹ , O, S, NR ¹⁹ with the proviso that for formulas (VIII) and (IX), when Z = CH, X is O or S ;
R ¹⁹ is, H, alkyl, cycloalkyl, aryl, heteroaryl, - oxyalkyl, - oxyaryl, carbonyl, amido, hydroxy, selected from the group consisting of NO _2, NH _2, CN;
R ²⁰ and R ²¹ are independently selected from H, alkyl, cycloalkyl, heterocycle, aryl, heteroaryl, -oxyalkyl, -oxyaryl, carbonyl, amide, NO ₂ , NH ₂ , CN, CF ₃ Is;
X ¹ , X ² , and X ³ are independently O or S, provided that X ² and X ³ are not both O;
Y is O or S, provided that Y may not be O when the carbocyclic ring formed by R ¹⁷ and R ¹⁸ has 3 members in the ring;
Z is CH or N;
R ¹¹ , R ¹² , R ¹³ , and R ¹⁴ are independently H, alkyl chain, alkenyl chain, alkynyl chain, cycloalkyl, carbocycle, aryl, heteroaryl, heterocycle, halogen, alkoxy-, -thioalkyl, May be selected from carboxyl, carboxylic ester, carbonyl, carbamide, carbimide, thiocarbamide, or thiocarbonyl, NH ₂ , NO ₂ ;
R ¹⁵ and R ¹⁶ are each independently H, or a branched or unbranched alkyl chain, or a branched or unbranched alkenyl chain;
R ¹⁷ and R ¹⁸ are independently selected from H or an alkyl chain, alkenyl chain, alkynyl chain, carbocycle, aryl, heteroaryl, heteroalkyl, or so as to form a carbocycle with up to 6 ring atoms. Can be bound to;
n is 0 or 1.

は、式1から除外される。 In one condition, the following compound:

Is excluded from Equation 1.

A is an alkyl chain, alkenyl chain, or when it is selected from alkynyl chain, preferably, A is a C ₁ -C ₇ alkyl chain, C ₁ -C ₇ alkenyl chain, or C ₁ -C ₇ alkynyl chain . In one embodiment of the invention A is an unbranched C _2-5 alkyl chain, in particular an unbranched C _3-4 alkyl chain, in particular an unbranched C ₃ alkyl chain. In a second embodiment of the invention, A represents a C ₃ alkyl chain substituted at two positions by one (ie in S or R configuration) or by two methyl groups.

When A is selected from formulas (I) to (V), preferably A is selected from the groups (I) to (IV). In one embodiment of the invention, A represents a group of formula (IV), wherein n ¹ is each equal to 1 and m = 1-4, in particular m = 1. In a second embodiment of the present invention A represents a group of formula (I), (II) or (III), wherein n and n ¹ are each equal to 1, R ⁶ , R ⁷ , R ⁸ , R ⁹ , and R ¹⁰ represent H.

Preferably, R ⁶ , R ⁷ , R ⁸ , R ⁹ , and R ¹⁰ represent H or methyl.

  In one embodiment of the invention, the group B is from (VI), (VIa), (VIb), (VII), (X), (XI), (XII), (XIII), and (XIV) Selected. In a second embodiment of the invention, the group B represents formula (VI). In a third embodiment of the invention, the group B represents formula (VIa). In a fourth embodiment of the invention, the group B represents formula (VIb). In a fifth embodiment of the invention, the group B represents formula (VII). In a sixth embodiment of the invention, the group B represents formula (X). In a seventh embodiment of the invention, the group B represents formula (XI). In an eighth embodiment of the invention, the group B represents formula (XII). In another embodiment of the inventive group, the group B represents formula (XIII). In a further embodiment of the invention the group B represents formula (XIV). In a preferred embodiment of the invention B represents a group of formula (VI) or (VII).

  When B suitably represents the group (IX), A does not represent alkynyl.

  Preferably, D and E independently represent benzyl, aryl, heteroaryl or heterocycle.

  In one embodiment of the invention, D and E represent aryl, in particular phenyl, or naphthyl, in particular substituted phenyl. Preferred substituents when D represents phenyl include alkoxy-, -thioalkyl, halogen, or alkyl carboxylates or aryl esters. Also preferably, fluoro, chloro, bromo, iodo, trifluoromethyl, trifluoromethoxy, methoxy, ethoxy, benzyloxy, cyano, acetyl, dimethylamino, methylsulfanyl, nitro, oxazolyl, pyrazolyl, isopropyl, ethyl, and carbo Methoxyl. When the phenyl group is monosubstituted, the substitution is preferably in the 4-position. Other suitable aryl groups that D and E may represent include dihydrobenzodioxin, benzodioxole, benzodithiol dihydrobenzodithiine, benzooxathiol, and dihydrobenzooxathiine Including. A particularly preferred group that D or E may represent is 3,4- (dimethoxy) -phenyl.

Preferably, R ²⁰ and R ²¹ represent NO ₂ , CN, CF ₃ , or when R ²⁰ is H, R ²¹ is NO ₂ , CN, CF ₃ , or R ²¹ is When it is H, R ²⁰ is NO ₂ , CN, CF ₃ .

In one embodiment, X or Y is S, O, or NR ¹ . Preferably, X or Y is S.

  Preferably Z represents N.

In a preferred embodiment, R ¹¹ and R ¹⁴ are H.

In a further preferred embodiment, R ¹² and R ¹³ are independently selected from oxyalkyl, or thioalkyl, halogen, or carboxylic acid alkyl ester, or phenyl.

In a preferred embodiment, at least one of R ¹⁵ and R ¹⁶ is H, more preferably R ¹⁵ and R ¹⁶ are both H.

In a preferred embodiment, one of R ¹⁷ and R ¹⁸ is H and the other is Me. Preferably, one of R ¹⁷ and R ¹⁸ is H and the other is phenyl. In addition, preferred are compounds in which R ¹⁷ and R ¹⁸ form a carbocyclic ring having up to 6 members in the ring atoms.

  Preferred compounds include those defined by Examples 13, 119, and 125 below.

は、式1から除外される。 The present invention provides a compound of formula 1 for use as a medicament. In one embodiment, for the use of a compound of formula 1 as a medicament, the compound:

Is excluded from Equation 1.

  Compound (a) under the above conditions is disclosed as Compound 7 in Ganellin et al. (1995) J Med Chem 38 (17) 3342-3350. This article discloses the compounds as weak inhibitors of histamine H3 receptor.

  The compound of condition (b) is disclosed as compound 7 in Venkatachalam et al. (2001) Bioorganic Med Chem Lett 11, 523-528. This discloses the compound as an HIV1 reverse transcriptase inhibitor.

  The compound of condition (c) is disclosed as compound 19b in Moon et al. (1991) J Med Chem 34, 231-2327. This paper discloses the compounds as cholinergic agonists with potential use in the treatment of Alzheimer's disease.

  Compounds of condition (d) are disclosed as compounds 99, 100, and 102-103 in Wright et al. (1986) J Med Chem 29, 523-530. This article discloses the compounds as thromoxane synthetase inhibitors.

Certain compounds that would be encompassed by Formula 1 are those described in Wright et al. (1987) J Med Chem, unless there is a requirement that “X ² and X ³ are not both O”. 30, 2277-2283, disclosed as thromboxane synthetase inhibitors.

Certain compounds that will be encompassed by Formula 1 are those under the condition that "when the carbocycle formed by R ¹⁷ and R ¹⁸ has 3 members in the ring, Y may not be O" If not, it is disclosed in EP 0 117 462 A2 as a thromboxane synthetase inhibitor.

In particular:
Suitable compounds, shown below for formula 1 *, are inhibitors of QC:

式中、Rは、実施例1〜53において定義してある。

In a further embodiment, the inhibitor of QC (EC) is of formula 1a:

Wherein R is defined in Examples 1-53.

式中、R¹、及びR²は、実施例54〜95において定義してある。

Further suitable inhibitors of QC (EC) are those of formula 1b

Wherein R ¹ and R ² are defined in Examples 54-95.

式中、R³は、実施例96〜102において定義してある。

Further suitable inhibitors of QC (EC) are those of formula 1c

Wherein R ³ is defined in Examples 96-102.

式中、環上の位置は、実施例103〜105において定義してある。

Further suitable inhibitors of QC (EC) are those of formula 1d

Where the position on the ring is defined in Examples 103-105.

式中
R⁴、及びR⁵は、実施例106〜109において定義してある。

Further suitable inhibitors of QC (EC) are those of formula 1e

In the formula
R ⁴ and R ⁵ are defined in Examples 106-109.

式中
R⁶は、実施例110〜112において定義してある。

Further suitable inhibitors of QC (EC) are those of formula 1f

In the formula
R ⁶ is defined in Examples 110-112.

式中
R⁷、R⁸、及びR⁹は、実施例113〜132において定義してある。

Further suitable inhibitors of QC (EC) are those of formula 1g

In the formula
R ⁷ , R ⁸ , and R ⁹ are defined in Examples 113-132.

式中、nは、実施例133〜135において定義してある。

Further suitable inhibitors of QC (EC) are those of formula 1h

Where n is defined in Examples 133-135.

式中、mは、実施例136、及び137において定義してある。

Further suitable inhibitors of QC (EC) are those of formula 1i

Where m is defined in Examples 136 and 137.

Further suitable inhibitors of QC (EC) are those of formulas 138-141.

Preferred inhibitors of glutaminyl peptide cyclotransferase are 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride (also named QCI) It is.

  In a preferred embodiment, the present invention provides at least one QC inhibitor, optionally a nootropic, neuroprotective, antiparkinsonian, amyloid protein deposition inhibitor, beta amyloid synthesis inhibitor, antidepressant, anxiolytic Provided is a composition, preferably a pharmaceutical composition, comprising in combination with at least one other drug selected from the group consisting of a drug, an antipsychotic drug, and an anti-multiple sclerosis drug.

  More specifically, the other drugs mentioned above are selected from the group consisting of: β-amyloid antibody, cysteine protease inhibitor, PEP-inhibitor, LiCl, acetylcholinesterase (AChE) inhibitor, PIMT enhancer, β-secretase inhibitor, γ-secretase inhibitor, neutral endopeptidase inhibitor, phosphodiesterase-4 (PDE-4) inhibitor, TNFα inhibitor, muscarinic M1 receptor antagonist, NMDA receptor antagonist, Sigma-1 Receptor inhibitors, histamine H3 antagonists, immunomodulators, immunosuppressants, MCP-1 antagonists, or antegren (Natalizumab), Neurelan (Fampridine-SR), campath (Alemtuzumab), IR 208, NBI 5788 / MSP 771 (tiplimotide), paclitaxel, anelix .MS (Anergix.MS) (AG 284), SH636, Differin (CD 271, Adaprene), BAY 361677 (Interleukin-4), Matrix-metalloproteinase inhibitors (eg BB 76163), Interferon-τ (Trophoblastine) and a drug selected from the group consisting of SAIK-MS.

Furthermore, the other drug may be, for example, an anxiolytic or antidepressant selected from the group consisting of:
(A) benzodiazepines such as alprazolam, chlordiazepoxide, clobazam, clonazepam, chlorazepate, diazepam, fludiazepam, loflazepate, lorazepam, metacarone, oxazepam, prazepam, traxene,
(B) selective serotonin reuptake inhibitors (SSRI) such as citalopram, fluoxetine, fluvoxamine, escitalopram, sertraline, paroxetine,
(C) tricyclic antidepressants such as amitriptyline, clomipramine, desipramine, doxepin, imipramine,
(D) monoamine oxidase (MAO) inhibitors,
(E) Azapirones, such as buspirone, tandopsirone,
(F) a serotonin-norepinephrine reuptake inhibitor (SNRI), such as venlafaxine, duloxetine,
(G) Miltrazapine,
(H) a norepinephrine reuptake inhibitor (NRI), such as reboxetine,
(I) bupropion,
(J) nefazodone,
(K) β-blocker,
(L) NPY-receptor ligand: NPY agonist or antagonist.

In further embodiments, the other agent may be, for example, an anti-multiple sclerosis drug selected from the group consisting of:
a) dihydroorotic acid dehydrogenase inhibitors such as SC-12267, teriflunomide, MNA-715, HMR-1279 (syn. to HMR-1715, MNA-279),
b) autoimmune suppressants such as laquinimod,
c) paclitaxel,
d) Antibodies such as AGT-1, anti-granulocyte-macrophage colony stimulating factor (GM-CSF) monoclonal antibody, Nogo receptor modulator, ABT-874, alemtuzumab (CAMPATH), anti-OX40 antibody, CNTO-1275, DN-1921 , Natalizumab (syn. To AN-100226, Antegren, VLA-4 Mab), Daclizumab (syn. To Genepax) Ro-34-7375, SMART Anti-Tac, J-695, Priliximab (priliximab) ( syn. to Centara, CEN-000029, cM-T412), MRA, Dantes, anti-IL-12-antibody,
e) peptide nucleic acid (PNA) formulations, such as reticulose,
f) interferon alpha, such as alpha feron (Alfaferone), human alpha interferon (syn. to Omniferon, alpha leukoferon)
g) Interferon β, eg, Frone, interferon β-1a such as Avonex, Betron (Rebif), interferon β analog, interferon β-transferrin fusion protein, betaseron Recombinant interferon β-1b,
h) Interferon τ,
i) Peptides such as cyclic peptides such as AT-008, AnergiX.MS, Immunokine (α-immunokine-NNSO3), ZD-7349,
j) therapeutic enzymes such as soluble CD8 (sCD8),
k) a plasmid encoding multiple sclerosis-specific self-antigen and a plasmid encoding a cytokine, such as BHT-3009;
l) Inhibitors of TNFα, such as BLX-1002, thalidomide, SH-636,
m) TNF antagonists such as solimastat, lenercept (syn. to RO-45-2081, Tenefuse), onercept (sTNFR1), CC-1069
n) TNFα, eg etanercept (syn. to emblem, TNR-001)
o) CD28 antagonists such as abatacept,
p) Lck tyrosine kinase inhibitor,
q) Cathepsin K inhibitors,
r) Neuron-targeting membrane transport protein taurine, and analogs of the plant-derived calpain inhibitor leupeptin, such as Neurodur,
s) Chemokine receptor-1 (CCR1) antagonists such as BX-471,
t) CCR2 antagonist,
u) AMPA receptor antagonists such as ER-167288-01 and ER-099487, E-2007, talampanel,
v) Potassium channel blockers such as fampridine,
w) Tosyl-proline-phenylalanine small molecule antagonist of VLA-4 / VCAM interaction, eg TBC-3342
x) cell adhesion molecule inhibitors such as TBC-772,
y) antisense oligonucleotides such as EN-101,
z) an antagonist of a free immunoglobulin light chain (IgLC) that binds to the mast cell receptor, eg F-991,
aa) an apoptosis-inducing antigen, such as Apogen MS,
bb) alpha-2 adrenergic receptor agonists such as tizanidine (syn. to Zanaflex, Ternerin, Sirdalvo, Sirdalud, Minionidine),
cc) copolymers of L-tyrosine, L-lysine, L-glutamic acid, and L-alanine, such as glatiramer acetate (syn. to Copaxone, COP-1, copolymer-1),
dd) topoisomerase II modulators, such as mitoxantrone hydrochloride,
ee) adenosine deaminase inhibitors such as cladribine (syn. to Leustatin, Mylinax, RWJ-26251)
ff) Interleukin-10, such as ilodecakin (syn. to Tenovil, Sch-52000, CSIF),
gg) interleukin-12 antagonists such as lysofylline (syn. to CT-1501R, LSF, lysofylline),
hh) Ethanaminum, eg SRI-62-834 (syn. to CRC-8605, NSC-614383)
ii) immunomodulators such as SAIK-MS, PNU-156804, α-fetoprotein peptide (AFP), IPDS,
jj) Retinoid receptor agonists such as adaprene (syn. to Differin, CD-271),
kk) TGF-β, eg GDF-1 (growth and differentiation factor 1),
ll) TGF-β-2, such as BetaKine,
mm) MMP inhibitors such as glycomed,
nn) phosphodiesterase 4 (PDE4) inhibitors, such as RPR-122818,
oo) Purine nucleoside phosphorylase inhibitors such as 9- (3-pyridylmethyl) -9-deazaguanine, perdecin (syn. to BCX-34, TO-200)
pp) α-4 / β-1 integrin antagonists such as ISIS-104278,
qq) antisense α4 integrin (CD49d), eg ISIS-17044, ISIS-27104,
rr) cytokine inducers such as nucleosides, ICN-17261,
ss) a cytokine inhibitor,
tt) heat shock protein vaccine, eg HSPPC-96,
uu) Neuregulin growth factor, eg GGF-2 (syn. to neuregulin, glial growth factor 2)
vv) cathepsin S-inhibitors,
ww) bropirimine analogues such as PNU-56169, PNU-63693,
xx) Monocyte chemoattractant protein-1 inhibitors, such as benzimidazole derivative-like MCP-1 inhibitors, LKS-1456, PD-064036, PD-064126, PD-084486, PD-172084, PD-172386.

  Furthermore, the present invention provides a pharmaceutical composition, eg for parenteral, enteral or oral administration, comprising at least one QC inhibitor, optionally in combination with at least one other aforementioned agent. To do.

  These combinations provide a particularly beneficial effect. Therefore, such a combination is shown to be effective and useful for the treatment of the diseases mentioned above. Accordingly, the present invention provides a method for the treatment of these conditions.

  The method includes any simultaneous administration of at least one QC inhibitor and at least one other agent, or sequential administration thereof.

  Co-administration includes administration of a formulation comprising at least one QC inhibitor and at least one of the other agents, or essentially simultaneous administration of separate formulations of each agent.

に記述されている。 Β-amyloid antibodies and compositions containing the same include, for example,

It is described in.

  The β-amyloid antibody may be selected from, for example, polyclonal, monoclonal, chimeric, or humanized antibodies. Furthermore, the antibodies will be useful for developing active and passive immunotherapy (ie vaccines and monoclonal antibodies).

  Suitable examples of β-amyloid antibodies are ACU-5A5, huC091 (Acumen / Merck); PF-4360365, RI-1014, RI-1219, RI-409, RN-1219 (Rinat Neuroscience (Pfizer Inc)); Ablynx / Boehringer Ingelheim nanobody treatment; Intellect Neurosciences / IBL β-amyloid-specific humanized monoclonal antibody; m266, m266.2 (Eli Lilly &Co.); AAB-02 (Elan); bapineuzumab (bapineuzumab) ( Elan); BAN-2401 (Bioarctic Neuroscience AB); ABP-102 (Abiogen Pharma SpA); BA-27, BC-05 (Takeda); R-1450 (Roche); ESBA-212 (ESBATech AG); AZD-3102 (AstraZeneca), and Mindset BioPharmaceuticals Inc. β-amyloid antibody.

  Particularly preferred is an antibody that recognizes the N-terminus of the Aβ peptide. A suitable antibody that recognizes the Aβ-N-terminus is, for example, Acl-24 (AC immune CA). Monoclonal antibodies against β-amyloid peptide are disclosed in WO 2007/068412. Each chimeric and humanized antibody is disclosed in WO 2008/011348. A method for producing a vaccine composition for treating amyloid-related diseases is disclosed in WO 2007/068411.

に記述されている。 Suitable cysteine protease inhibitors are, for example, inhibitors of cathepsin B. Inhibitors of cathepsin B, and compositions comprising such inhibitors include, for example:

It is described in.

  An example of a suitable PIMT enhancer is 10-aminoaliphatyl-dibenz [b, f] oxepin, described in WO 98/15647 and WO 03/057204, respectively. The modulators of PIMT activity described in WO 2004/039773 are further useful in the present invention.

に記述されている。 Inhibitors of β-secretase and compositions containing such inhibitors include, for example:

It is described in.

  Suitable examples of β-secretase inhibitors for the present invention include WY-25105 (Wyeth); Posiphen, (+)-phenserine (TorreyPines / NIH); LSN-2434074, LY-2070275, LY-2070273, LY-2070102 (Eli Lilly &Co.); PNU-159775A, PNU-178025A, PNU-17820A, PNU-33312, PNU-38773, PNU-90530 (Elan / Pfizer); KMI-370, KMI-358, kmi- 008 (Kyoto University); OM-99-2, OM-003 (Athenagen Inc.); AZ-12304146 (AstraZeneca / Astex); GW-840736X (GlaxoSmithKline plc.), And DNP-004089 (De Novo Pharmaceuticals Ltd.) .

に記述されている。 Inhibitors of gamma secretase and compositions containing such inhibitors include, for example:

It is described in.

  Suitable γ-secretase inhibitors for the purposes of the present invention include GSI-953, WAY-GSI-A, WAY-GSI-B (Wyeth); MK-0752, MRK-560, L-852505, L-685- 458, L-852631, L-852646 (Merck & Co. Inc.); LY-450139, LY-411575, AN-37124 (Eli Lilly &Co.); BMS-299897, BMS-433796 (Bristol-Myers Squibb Co) E-2012 (Eisai Co. Ltd.); EHT-0206, EHT-206 (ExonHit Therapeutics SA); and NGX-555 (TorreyPines Therapeutics Inc.).

  Suitable beta amyloid synthesis inhibitors for the purposes of the present invention are, for example, Bisnorcymserine (Axonyx Inc.); (R) -flurbiprofen (MCP-7869; Flurizan) (Myriad Genetic); nitroflurbi Profen (NicOx); BGC-20-0406 (Sankyo Co. Ltd.) and BGC-20-0466 (BTG plc.).

  Suitable amyloid protein deposition inhibitors for the purposes of the present invention include, for example, SP-233 (Samaritan Pharmaceuticals); AZD-103 (Ellipsis Neurotherapeutics Inc.); AAB-001 (Bapineuzumab), AAB-002, ACC-001 ( Elan Corp plc.); Colostrinin (ReGen Therapeutics plc.); Tramiprosate (Neurochem); AdPEDI- (amyloid-β1-6) 11) (Vaxin Inc.); MPI-127585, MPI-423948 (Mayo Foundation) SP-08 (Georgetown University); ACU-5A5 (Acumen / Merck); Transthyretin (State University of New York); PTI-777, DP-74, DP 68, Exebryl (ProteoTech Inc.); m266 (Eli) Lilly &Co.); EGb-761 (Dr. Willmar Schwabe GmbH); SPI-014 (Satori Pharmaceuticals Inc.); ALS-633, ALS-499 (Advanced Life Sciences Inc.); AGT-160 (ArmaGen Technologies Inc.) ); TAK-070 (Takeda Pharmaceutical Co. Ltd.); CHF-5022, CHF-5074, CHF-5096, and CHF-5105 (Chiesi Farmaceutici SpA.).

Suitable PDE-4 inhibitors for the purposes of the present invention are, for example, Doxofylline (Instituto Biologico Chemioterapica ABC SpA.); Iddilast ophthalmic, tipelukast, ibudilast (Kyorin Pharmaceutical Co. Ltd.) Theophylline (Elan Corp.); cilomilast (GlaxoSmithKline plc.); Atopik (Barrier Therapeutics Inc.); tofimilast (CI-1044, PD-189659, CP-220629, PDE 4d inhibitor) BHN (Pfizer Inc.); allophylline, LAS-37779 (Almirall Prodesfarma SA.); Roflumilast, hydroxypumafentrine (Altana AG), tetomilast (Otska Pharmaceutical Co. Ltd. ); Tipelukast, ibudilast (Kyorin Pharmaceutical), CC-10004 (Celgene Corp.); HT-0712, IPL-4088 (Inflazyme Pharmaceutica) ls Ltd.); MEM-1414, MEM-1917 (Memory Pharmaceuticals Corp.); oglemilast, GRC-4039 (Glenmark Pharmaceuticals Ltd.); AWD-12-281, ELB-353, ELB-526 (Elbion AG) ); EHT-0202 (ExonHit Therapeutics SA.); ND-1251 (Neuro3d SA.); 4AZA-PDE4 (4 AZA Bioscience NV); AVE-8112 (Sanofi-Aventis); CR-3465 (Rottapharm SpA.); GP-0203, NCS-613 (Centre National de la Recherche Scientifique); KF-19514 (Kyowa Hakko Kogyo Co. Ltd.); Ono-6126 (Ono Pharmaceutical Co. Ltd.); OS-0217 (Dainippon Pharmaceutical Co. Ltd.) IBFB-130011, IBFB-150007, IBFB-130020, IBFB-140301 (IBFB Pharma GmbH); IC-485 (ICOS Corp.); RBx-14016, and RBx-11082 (Ranbaxy ory Ltd.). A preferred PDE-4-inhibitor is Rolipram.

に記述されている。 MAO inhibitors and compositions containing such inhibitors include, for example:

It is described in.

  Suitable MAO inhibitors for the purposes of the present invention are, for example, Linezolid (Pharmacia Corp.); RWJ-416457 (RW Johnson Pharmaceutical Research Institute); budapine (Altana AG); GPX-325 (BioResearch) Ireland); isocarboxazide; phenelzine; tranylcypromine; indantadol (Chiesi Farmaceutici SpA.); Moclobemide (Roche Holding AG); SL-25.1131 (Sanofi-Synthelabo); CX-1370 (Burroughs Wellcome Co. CX-157 (Krenitsky Pharmaceuticals Inc.); Desoxypeganine (HF Arzneimittelforschung GmbH & Co. KG); Bifemelan (Mitsubishi-Tokyo Pharmaceuticals Inc.); RS-1636 (Sankyo Co. Ltd.); Esuprone (BASF AG); rasagiline (Teva Pharmaceutical Industries Ltd.); ladostigil (Hebrew University of Jerusalem); safinamide (Pfi) zer), and NW-1048 (Newron Pharmaceuticals SpA.).

  Suitable histamine H3 antagonists for the purposes of the present invention include, for example, ABT-239, ABT-834 (Abbott Laboratories); 3874-H1 (Aventis Pharma); UCL-2173 (Berlin Free University), UCL-1470 (BioProjet, Societe Civile de Recherche); DWP-302 (Daewoong Pharmaceutical Co Ltd); GSK-189254A, GSK-207040A (GlaxoSmithKline Inc.); Ciprarisant (cipralisant), GT-2203 (Gliatech Inc.); Siproxifan (Ciproxifan) INSERM), 1S, 2S) -2- (2-aminoethyl) -1- (1H-imidazol-4-yl) cyclopropane (Hokkaido University); JNJ-17216498, JNJ-5207852 (Johnson &Johnson); NNC- 0038-0000-1049 (Novo Nordisk A / S); and Sch-79687 (Schering-Plough).

に記述されている。 PEP inhibitors and compositions containing such inhibitors include, for example,

It is described in.

  Suitable prolyl endopeptidase inhibitors for the purposes of the present invention include, for example, Fmoc-Ala-Pyrr-CN, Z-Phe-Pro-benzothiazole (Probiodrug), Z-321 (Zeria Pharmaceutical Co Ltd.); ONO -1603 (Ono Pharmaceutical Co Ltd); JTP-4819 (Japan Tobacco Inc.), and S-17092 (Servier).

  Other suitable compounds that can be used in accordance with the present invention in combination with a QC inhibitor are NPY, NPY mimetics, or NPY agonists or antagonists, or ligands for NPY receptors.

  NPY receptor antagonists are preferred in the present invention.

  Suitable ligands or antagonists of the NPY receptor are compounds derived from 3a, 4,5,9b-tetrahydro-1H-benz [e] indol-2-ylamine as disclosed in WO 00/68197.

に開示されたものを含む。好ましいNPYアンタゴニストは、これらの特許文献に具体的に開示されている化合物を含む。より好ましい化合物は、アミノ酸、及び非ペプチドに基づいたNPYアンタゴニストを含む。言及してもよいアミノ酸、及び非ペプチドに基づいたNPYアンタゴニストは、

に開示されたものを含む。好ましいアミノ酸、及び非ペプチドに基づいたNPYアンタゴニストは、これらの特許文献に具体的に開示されている化合物を含む。 NPY receptor antagonists that may be mentioned are:

Including those disclosed in. Preferred NPY antagonists include those compounds specifically disclosed in these patent documents. More preferred compounds include amino acid and non-peptide based NPY antagonists. NPY antagonists based on amino acids and non-peptides that may be mentioned are

Including those disclosed in. Preferred amino acid and non-peptide based NPY antagonists include those compounds specifically disclosed in these patent documents.

  Particularly preferred compounds include NPY antagonists based on amino acids. Compounds based on amino acids that may be mentioned include those disclosed in International Patent Application Publication Nos. WO 94/17035, WO97 / 19911, WO97 / 19913, WO97 / 19914, or preferably WO99 / 15498. Preferred amino acid-based NPY antagonists are those specifically disclosed in these patent documents, such as BIBP 3226, and in particular (R) -N2- (diphenylacetyl)-(R) -N- [1- ( 4-hydroxy-phenyl) ethyl] argininamide (Example 4 of International Patent Application Publication No. WO99 / 15498).

  M1 receptor agonists and compositions comprising such inhibitors are described, for example, in WO2004 / 087158, WO91 / 10664.

  Suitable M1 receptor antagonists for the purposes of the present invention include, for example, CDD-0102 (Cognitive Pharmaceuticals); Cevimeline (Evoxac) (Snow Brand Milk Products Co. Ltd.); NGX-267 (TorreyPines Therapeutics); Sabcomeline (GlaxoSmithKline); alvameline (H Lundbeck A / S); LY-593093 (Eli Lilly &Co.); VRTX-3 (Vertex Pharmaceuticals Inc.); WAY-132983 (Wyeth), and CI -101 7 / (PD-151832) (Pfizer Inc.).

に記述されている。 Acetylcholinesterase inhibitors and compositions containing such inhibitors include, for example:

It is described in.

  Suitable acetylcholinesterase inhibitors for the purposes of the present invention are, for example, Donepezil (Eisai Co. Ltd.); Rivastigmine (Novartis AG); (-)-Phenserine (TorreyPines Therapeutics); Ladostigil (Hebrew) University of Jerusalem); hyperzine A (Mayo Foundation); Galantamine (Johnson &Johnson); Memoquin (Universita di Bologna); SP-004 (Samaritan Pharmaceuticals Inc.); BGC-20-1259 (Sankyo Co.) Physostigmine (Forest Laboratories Inc.); NP-0361 (Neuropharma SA); ZT-1 (Debiopharm); Tacrine (Warner-Lambert Co.); Metriphonate (Bayer Corp.) and INM-176 (WhanIn ).

に記述されている。 NMDA receptor antagonists and compositions comprising such inhibitors include, for example:

It is described in.

  Suitable NMDA receptor antagonists for the purposes of the present invention include, for example, Memantine (Merz & Co. GmbH); Topiramate (Johnson &Johnson); AVP-923 (Neurodex) (Center for Neurologic Study); EN- 3231 (Endo Pharmaceuticals Holdings Inc.); neramexane (MRZ-2 / 579) (Merz and Forest); CNS-5161 (CeNeS Pharmaceuticals Inc.); dexanabinol (HU-211; sinnavidol) (Sinnabidol); PA-50211) (Pharmos); EpiCept NP-1 (Dalhousie University); Indantadol (V-3381; CNP-3381) (Vernalis); Perzinfotel (EAA-090, WAY- 126090, EAA-129) (Wyeth); RGH-896 (Gedeon Richter Ltd.); traxoprodil (CP-101606), besonprodil (PD-196860, CI-1041) (Pfizer Inc.); CGX -1007 (Cognetix Inc.); delucemine (NPS-1506) (NPS Pharmaceuticals Inc.); EVT-101 (Roche Holding AG); (Synchroneuron LLC.); Acamprosate, CR-3991, CR-2249, CR-3394 (Rottapharm SpA.); AV-101 (4- Cl-kynurenine (4-Cl-KYN)), 7-chloro-kynurenic acid (7-Cl-KYNA) (VistaGen); NPS-1407 (NPS Pharmaceuticals Inc.); YT-1006 (Yaupon Therapeutics Inc.); ED -1812 (Sosei R & D Ltd.); himantane (N-2- (adamantri) -hexamethylene-imine hydrochloride (RAMS); Lancicemine (AR-R-15896) (AstraZeneca); EVT-102 Ro-25-6981 and Ro-63-1908 (Hoffmann-La Roche Ag / Evotec).

  Furthermore, the present invention administers a QC inhibitor in combination with another therapeutic agent selected from the group consisting of the following, providing beneficial or synergistic therapeutic efficacy over each monotherapy component alone: Concerning combination therapies useful for the treatment of atherosclerosis, restenosis, pancreatitis, or arthritis: inhibitors of angiotensin converting enzyme (ACE); angiotensin II receptor blockers; diuretics; calcium channel blockers (CCB) Β-blockers; platelet aggregation inhibitors; cholesterol absorption modulators; HMG-Co-A reductase inhibitors; compounds that increase high density lipoprotein (HDL); renin inhibitors; IL-6 inhibitors; Corticosteroids; antiproliferative drugs; nitric oxide donors; inhibitors of extracellular matrix synthesis; growth factors or cytokine signaling inhibitors; MCP-1 antagonists And tyrosine kinase inhibitors.

  It is understood that an angiotensin II receptor blocker is an active agent that binds to the AT1-receptor subtype of angiotensin II receptor but does not cause receptor activation. As a result of the blockade of the AT1 receptor, these antagonists can be used, for example, as antihypertensive drugs.

の名称E-41 77
以下の式

の名称SC-52458をもつ化合物、
及び式

の名称ZD-8731をもつ化合物、
又はいずれの場合においても、これらの医薬として許容し得る塩。 Suitable angiotensin II receptor blockers that may be used in the combinations of the present invention include AT1 receptor antagonists having different structural characteristics, preferably those having a non-peptidic structure. For example, reference may be made to a compound selected from the group consisting of: Valsartan (EP 443983), Losartan (EP 253310), Candesartan (EP 459136), Eprosartan (EP 403159), Irbesartan (EP 454511) Olmesartan (EP 503785), Tasosartan (EP 539086), Telmisartan (EP 522314), formula

Name of E-41 77
The following formula

A compound having the name SC-52458,
And formula

A compound having the name ZD-8731,
Or, in any case, these pharmaceutically acceptable salts.

  A preferred AT1-receptor antagonist is an approved and marketed drug, most preferably valsartan, or a pharmaceutically acceptable salt thereof.

  Interfering enzymatic degradation of angiotensin with angiotensin II by ACE inhibitors is a good variant for the control of blood pressure and can therefore be used in therapeutic methods for the treatment of hypertension.

  Suitable ACE inhibitors for use in the combinations of the present invention are, for example, compounds selected from: alacepril, benazepril, benazeprilate, captopril, ceronapril, cilazapril, delapril, enalapril, enaprilat, fosinopril (Fosinopril), imidapril, lisinopril, movetopril, perindopril, quinapril, ramipril, spirapril, temocapril, and trandolapril, or in each case, a pharmaceutically acceptable salt thereof.

  Preferred ACE inhibitors are commercially available drugs, most preferably benazepril and enalapril.

  The diuretic is, for example, a thiazide derivative selected from the group consisting of chlorothiazide, hydrochlorothiazide, methylchlorothiazide, and chlorosalidon. The most preferred diuretic is hydrochlorothiazide. Diuretics further include amiloride or potassium-sparing diuretics such as triameterine, or pharmaceutically acceptable salts thereof.

  CCB types include dihydropyridines (DHP) such as diltiazem-type and verapamil-type CCB, as well as non-DHP.

  A CCB useful for the combination is preferably a DHP representative selected from the group consisting of amlodipine, felodipine, ryosidine, isradipine, lacidipine, nicardipine, nifedipine, nigurdipine, nildipine, nimodipine, nisoldipine, nitrendipine, and nivaldipine. And preferably a non-DHP representative selected from the group consisting of flunarizine, prenylamine, diltiazem, fendiline, galopamil, mibefradil, anipamyl, thiapamil, and verapamil, and pharmaceutically acceptable salts in each case It is. All these CCBs are used therapeutically, for example as antihypertensives, antianginals or antiarrhythmic drugs.

  Preferred CCBs include amlodipine, diltiazem, isradipine, nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine, and verapamil, or a pharmaceutically acceptable salt thereof, for example, depending on the specific CCB. Particularly preferred as DHP is amlodipine, or a pharmaceutically acceptable salt thereof, particularly besylate. A particularly preferred representative of non-DHP is verapamil, or a pharmaceutically acceptable salt, especially the hydrochloride salt.

  Beta blockers suitable for use in the present invention include beta-adrenergic blockers (beta blockers) that compete with epinephrine for beta adrenergic receptors and prevent the action of epinephrine. Preferably, the beta blocker is selective for the beta receptor compared to the alpha adrenergic receptor and thus has no significant alpha-inhibitory effect. Suitable beta blockers include compounds selected from acebutolol, atenolol, betaxolol, bisoprolol, carteolol, carvedilol, esmolol, labetalol, metoprolol, nadolol, oxprenolol, penbutolol, pindolol, propranolol, sotalol, and timolol. Including. If the beta blocker is capable of forming an acid, or base, or otherwise a pharmaceutically acceptable salt, or prodrug, these forms are considered to be encompassed herein and the compound is It is understood that it may be administered in a free form, such as a physiologically hydrolyzable and acceptable ester, or in the form of a pharmaceutically acceptable salt or prodrug. For example, metoprolol is suitably administered as its tartrate salt and propranolol is suitably administered as its hydrochloride salt and the like.

  Platelet aggregation inhibitors include PLAVIX® (clopidogrel bisulfate), PLETAL® (cilostazol), and aspirin.

  Cholesterol absorption modulators include ZETIA (registered trademark) (ezetimibe) and KT6-971 (Kotobuki Pharmaceutical Co. Japan).

  HMG-Co-A reductase inhibitors (also called β-hydroxy-β-methylglutaryl-co-enzyme-A reductase inhibitors, or statins) are used to lower blood lipid levels, including cholesterol. It is understood that this is an active agent that can be obtained.

  Types of HMG-Co-A reductase inhibitors include compounds with different structural characteristics. For example, a compound selected from the group consisting of atorvastatin, cerivastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin, or a pharmaceutically acceptable salt in each case can be mentioned.

  Preferred HMG-Co-A reductase inhibitors are commercially available drugs, most preferably atorvastatin, pitavastatin, or simvastatin, or a pharmaceutically acceptable salt thereof.

  Compounds that increase HDL include, but are not limited to, cholesterol ester transfer protein (CETP) inhibitors. Examples of CETP inhibitors include JTT7O5 disclosed in Example 26 of US Pat. No. 6,426,365 issued July 30, 2002, and pharmaceutically acceptable salts thereof.

  Inhibition of interleukin-6 mediated inflammation is either indirectly through the control of endogenous cholesterol synthesis and isoprenoid depletion, or interleukin-6 inhibitor / antibody, interleukin-6 receptor inhibitor / antibody, interleukin Leukine-6 antisense oligonucleotide (ASON), gp130 protein inhibitor / antibody, tyrosine kinase inhibitor / antibody, serine / threonine kinase inhibitor / antibody, mitogen-activated protein (MAP) kinase inhibitor / antibody Phosphatidylinositol 3-kinase (PI3K) inhibitor / antibody, nuclear factor κB (NF-κB) inhibitor / antibody, IκB kinase (IKK) inhibitor / antibody, activated protein-1 (AP-1) inhibitor / Antibodies, STAT transcription factor inhibitors / antibodies, altered IL-6, IL-6, or IL-6 receptor partial peptides, or SOCS (of cytokine signaling Inhibitors) may be achieved by direct inhibition of signaling pathways utilizing proteins, PPARγ, and / or PPARβ / delta activators / ligands, or functional fragments thereof.

  A suitable anti-inflammatory corticosteroid is dexamethasone.

  Suitable antiproliferative drugs are cladribine, rapamycin, vincristine, and taxol.

  A suitable inhibitor of extracellular matrix synthesis is halofuginone.

  A suitable growth factor or cytokine signaling inhibitor is for example the ras inhibitor R115777.

  A suitable tyrosine kinase inhibitor is tyrophostin.

  Suitable renin inhibitors are for example described in WO2006 / 116435. A preferred renin inhibitor is aliskiren, preferably in its hemifumarate form.

  MCP-1 antagonists include, for example, anti-MCP-1 antibodies, preferably monoclonal or humanized monoclonal antibodies, MCP-1 expression inhibitors, CCR2-antagonists, TNF-α inhibitors, VCAM-1 gene expression inhibitors, and Selected from anti-C5a monoclonal antibodies.

に記述されている。 MCP-1 antagonists and compositions comprising such inhibitors include, for example:

It is described in.

  Suitable MCP-1 antagonists include, for example, C-243 (Telik Inc.); NOX-E36 (Noxxon Pharma AG); AP-761 (Actimis Pharmaceuticals Inc.); ABN-912, NIBR-177 (Novartis AG); CC -11006 (Celgene Corp.); SSR-150106 (Sanofi-Aventis); MLN-1202 (Millenium Pharmaceuticals Inc.); AGI-1067, AGIX-4207, AGI-1096 (AtherioGenics Inc.); PRS-211095, PRS- 211092 (Pharmos Corp.); anti-C5a monoclonal antibodies such as neutrazumab (G2 Therapies Ltd.); AZD-6942 (AstraZeneca plc.); 2-mercaptoimidazole (Johnson &Johnson); TEI-E00526, TEI -6122 (Deltagen); RS-504393 (Roche Holding AG); SB-282241, SB-380732, ADR-7 (GlaxoSmithKline); anti-MCP-1 monoclonal antibody (Johnson & Johnson).

  A combination of an MCP-1 antagonist and a QC inhibitor will generally be useful for the treatment of inflammatory diseases, including neurodegenerative diseases.

  A combination of an MCP-1 antagonist and a QC inhibitor is preferred for the treatment of Alzheimer's disease.

  Most preferably, the QC inhibitor is combined with one or more compounds selected from the following group: PF-4360365, m266, bapineuzumab, R-1450, Posiphen, (+) -Phenserine, MK-0752, LY-450139, E-2012, (R) -flurbiprofen, AZD-103, AAB-001 (bapinewumab), tramiprosate, EGb-761, TAK-070, Doxophilin, theophylline, cilomilast, tofimilast, toflumilast, tetomilast, tipelukast, ibudilast, HT-0712, MEM-1414, oglemilast, linezolid, budipine, isocarboxazide, fenelzine, indanilpromine indantadol), moclobemide, rasagiline, ladostigil, safinamide, ABT-239, ABT-834, GSK-18 9254A, Siproxifan, JNJ-17216498, Fmoc-Ala-Pyrr-CN, Z-Phe-Pro-benzothiazole, Z-321, Ono-1603, JTP-4819, S-17092, BIBP3226; (R ) -N2- (diphenylacetyl)-(R) -N- [1- (4-hydroxyphenyl) ethyl] argininamide, cevimeline, sabcomeline, (PD-151832), donepezil, rivastigmine, (-)- Phenserine, Radostigil, Galantamine, Tacrine, Metrifonate, Memantine, Topiramate, AVP-923, EN-3231, Neramexane, Valsartan, Benazepril, Enalapril, Hydrochlorothiazide, Amlodipine, Diltiazem, Nidrapin Nisoldipine, nitrendipine, verapamil, amlodipine, acebutolol, atenolol, betaxolol Bisoprolol, carteolol, carvedilol, esmolol, labetalol, metoprolol, nadolol, oxprenolol, penbutolol, pindolol, propranolol, sotalol, timolol, PLAVIX (registered trademark) (clopidogrel bisulfate), PLETAL (registered trademark) (cilostazol), Aspirin, ZETIA® (ezetimibe), and KT6-971, statin, atorvastatin, pitavastatin, or simvastatin; dexamethasone, cladribine, rapamycin, vincristine, taxol, aliskiren, C-243, ABN-912, SSR-150106, MLN -1202, and betaferon.

In particular, the following combinations are possible:
-QC inhibitors, especially QCI, in combination with atorvastatin for the treatment and / or prevention of atherosclerosis,
-QC inhibitors, especially QCI, in combination with immunosuppressive agents, preferably rapamycin, for the prevention and / or treatment of restenosis
-QC inhibitors, especially QCI, in combination with immunosuppressants, preferably paclitaxel, for the prevention and / or treatment of restenosis,
-QC inhibitors, in particular QCI, in combination with AChE inhibitors, preferably donepezil, for the prevention and / or treatment of Alzheimer's disease
-QC inhibitors, in particular QCI, in combination with interferons, preferably Aronex, for the prevention and / or treatment of multiple sclerosis,
-QC inhibitors, in particular QCI, in combination with interferons, preferably betaferon, for the prevention and / or treatment of multiple sclerosis,
-QC inhibitors, especially QCI, in combination with interferons, preferably Rebif, for the prevention and / or treatment of multiple sclerosis,
-QC inhibitors, especially QCI, in combination with Copaxone for the prevention and / or treatment of multiple sclerosis,
-QC inhibitors, especially QCI, in combination with dexamethasone for the prevention and / or treatment of restenosis
-QC inhibitors, in particular QCI, in combination with dexamethasone for the prevention and / or treatment of atherosclerosis
-QC inhibitors, especially QCI, in combination with dexamethasone, for the prevention and / or treatment of rheumatoid arthritis
A QC inhibitor, in particular a QCI, in combination with an HMG-Co-A-reductase inhibitor for the prevention and / or treatment of restenosis, wherein the HMG-Co-A-reductase inhibitor is atorvastatin, Selected from cerivastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin,
A QC inhibitor, in particular a QCI, in combination with an HMG-Co-A-reductase inhibitor for the prevention and / or treatment of atherosclerosis, said HMG-Co-A reductase inhibitor Selected from atorvastatin, cerivastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin,
A QC inhibitor, in particular a QCI, in combination with an HMG-Co-A-reductase inhibitor for the prevention and / or treatment of rheumatoid arthritis, wherein the HMG-Co-A-reductase inhibitor is atorvastatin , Cerivastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.

  Such combination therapies are particularly useful for neurodegeneration in AD, FAD, FDD, and Down's syndrome, and atherosclerosis, rheumatoid arthritis, restenosis, and pancreatitis.

  Such combination therapy may produce a superior therapeutic effect (less proliferation, as well as less inflammation, stimulation for proliferation) than would occur with either drug alone.

  See, in particular, in this regard, WO2004 / 098625, which is incorporated herein by reference, for specific QC inhibitor combinations with further compounds.

In a further embodiment, the present invention provides a method for preventing or treating a disease or condition selected from the group consisting of inflammatory diseases selected from:
a. neurodegenerative diseases such as mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down syndrome, familial British dementia, familial Danish dementia, multiple sclerosis,
b. Chronic and acute inflammation such as rheumatoid arthritis, atherosclerosis, restenosis, pancreatitis,
c. Fibrosis such as pulmonary fibrosis, liver fibrosis, kidney fibrosis,
d. Cancers such as cancer / angioendothelioma growth, gastric carcinoma,
e. Metabolic diseases such as hypertension,
f. and other inflammatory diseases such as neuropathic pain, graft rejection / graft failure / graft vasculopathy, HIV infection / AIDS, pregnancy toxemia, tuberous sclerosis.

  In addition, the present invention relates to the use of the compounds of the present invention and their corresponding pharmaceutically acceptable acid salt forms for the manufacture of a medicament for the prevention or treatment of any of the above mentioned diseases or conditions. Including.

  Most preferably, the QC inhibitor is used for the treatment of the aforementioned neurodegenerative diseases. More preferred is the use of a QC inhibitor of the present invention for the treatment of a disease selected from restenosis, pancreatitis, rheumatoid arthritis and atherosclerosis, most preferably restenosis or pancreatitis.

  The compound may be administered to the patient by any conventional route of administration, including but not limited to intravenous, oral, subcutaneous, intramuscular, intradermal, parenteral, and combinations thereof.

  In a further preferred form of implementation, the invention comprises a pharmaceutical composition comprising at least one compound of the invention, or a salt thereof, optionally in combination with one or more pharmaceutically acceptable carriers and / or solvents. Product, that is, medicine.

  The pharmaceutical composition may contain suitable carriers, for example parenterally or in the form of enteral preparations, or they contain suitable carriers suitable for oral administration. It may also be in the form of an oral formulation. Preferably these are in the form of an oral formulation.

  Inhibitors of QC activity administered in accordance with the present invention as inhibitors or in combination with inhibitors, substrates, pseudo-substrates, inhibitors of QC expression, binding proteins or enzyme protein antibodies that reduce QC protein concentration in mammals And may be used in pharmaceutical-administrable preparations or preparation complexes. The compounds of the present invention can be individually tailored to the patient and the disease, and in particular can avoid individual intolerance, allergies, and side effects.

  The compounds also exhibit different degrees of activity as a function of time. This gives the doctor providing the treatment the opportunity to respond differently to the individual situation of the patient: he exactly, on the one hand, the rate of onset of action, and on the one hand, the duration of action, and In particular, the strength of action can be adjusted.

  The compounds may conveniently be administered in the form of pharmaceutical preparations containing the active ingredients in combination with conventional additives such as diluents, excipients and / or carriers known from the prior art. . For example, they can be administered parenterally (eg, i.v. in physiological saline) or enterally (eg, orally, formulated with a conventional carrier).

  Depending on their intrinsic stability and their bioavailability, one or more compound doses can be given per day to achieve the desired reduction in MCP activity. For example, such a dose range in humans may be in the range of about 0.01 mg to 250.0 / mg per day, preferably about 0.01 to 100 mg of compound per kilogram of body weight per day.

  The compounds used according to the invention are per se known in the known manner, eg tablets, (biting) capsules, dragees, pills, suppositories, granules, aerosols, syrups, drops, liquids, solids and creams Inert, non-toxic, pharmaceutically suitable carriers, and additives or solvents in conventional formulations such as emulsions and suspensions and / or as suppositories or as nasal spray solutions Can be used and converted. In each of these formulations, the therapeutically effective compound is preferably sufficient to achieve the stated dosage tolerances, preferably at a concentration of approximately 0.1-80%, more preferably 1-50% by weight of the total mixture. Present in the correct amount.

  The formulations may be conveniently prepared, for example, by extending the active ingredient with a solvent and / or carrier, optionally using emulsifiers and / or dispersants, for example when water is used as the diluent. Organic solvents can optionally be used as auxiliary solvents.

  Examples of excipients useful in connection with the present invention include: water, paraffin (eg, natural oil fraction), vegetable oil (eg, rapeseed oil, peanut oil, sesame oil), alcohol (eg, ethyl alcohol, Glycerol), non-toxic organic solvents such as glycols (eg propylene glycol, polyethylene glycol); solid carriers, eg natural powdered minerals (eg highly dispersed silica, silicic acid), sugars (eg crude sugar, lactose Emulsifiers such as nonionic and anionic emulsifiers (eg, polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, alkyl sulfonates, and aryl sulfonates), dispersants (eg, lignin) , Sulfite cooking liquor, methylcellulose, starch, and polyvinylpyrroline Down), and lubricants (e.g., magnesium stearate, talc, stearic acid, and sodium lauryl sulphate) and optionally flavorings.

  Administration may be carried out in the usual manner, preferably enterally or parenterally, in particular orally. For enteral administration, tablets refer to additional additives such as sodium citrate, calcium carbonate, and calcium phosphate, along with various additives such as starch, preferably potato starch, gelatin and the like, in addition to the carriers mentioned. In addition to the above-mentioned carrier, it may be contained. Furthermore, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc can be used simultaneously for tableting. In the case of aqueous suspensions and / or elixirs intended for oral administration, various taste modifiers or colorants can be added to the active ingredient in addition to the excipients described above.

  For parenteral administration, a solution of the active ingredient can be used using a suitable liquid carrier. In general, for intravenous administration, it has been found advantageous to administer an amount of body weight of approximately 0.01 to 2.0 mg / kg, preferably approximately 0.01 to 1.0 mg / kg per day for effective results. For enteral administration, the dosage is approximately 0.01 to 2 mg / kg, preferably approximately 0.01 to 1 mg / kg body weight per day.

  Nevertheless, in some cases, depending on the body weight of the experimental animal or patient, or by the type of route of administration, but based on the species of animal and its individual response to the drug, or on the interval at which the administration is performed It may be necessary to deviate from the stated amounts. Thus, in some cases it may be sufficient to use less than the minimum amount mentioned above, but in other cases the upper limit mentioned may have to be exceeded. If relatively large amounts are administered, it may be desirable to divide these amounts into several single doses over the day. For administration in human medicine, the same dosage tolerances are provided. The above findings apply analogously to that case.

  The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following figures and examples. These examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Although specific terms have been used herein, such terms are intended in a descriptive sense and not for purposes of limitation.

(Reference Example 1: Preparation of human QC)
Host strains and media Pichia pastoris strain X33 (AOX1, AOX2) used for expression of human QC was cultured, transformed and analyzed according to the manufacturer's instructions (Invitrogen) . The media required for P. pastoris, buffered glycerol (BMGY) complex media or methanol (BMMY) complex media, and fermentation basal salt media were prepared according to the manufacturer's recommendations.

(Molecular cloning of plasmid vector encoding human QC)
All cloning procedures were performed applying standard molecular biology techniques. The vector pPICZαB (Invitrogen) was used for expression in yeast. Human QC was expressed in E. coli using the pQE-31 vector (Qiagen). The mature QC cDNA starting at codon 38 was fused in-frame with a plasmid encoding a 6x histidine tag. After amplification using primers pQCyc-1 and pQCyc-2 (WO2004 / 098625) and subcloning, the fragment was inserted into an expression vector using SphI and HindIII restriction sites.

(P. pastoris transformation and mini-scale expression)
Plasmid DNA was amplified in E. coli JM109 and purified according to manufacturer's (Qiagen) recommendations. In the expression plasmid used (pPICZαB), three restriction sites are provided for linearization. Since SacI and BstXI cleaved the cDNA in QC, PmeI was selected for linearization. 20-30 μg plasmid DNA was linearized with PmeI, precipitated with ethanol and dissolved in sterile deionized water. 10 μg of DNA was then applied for transformation of competent P. pastoris cells by electroporation according to the manufacturer's instructions (Biorad). Selection was performed using plates containing 150 μg / ml Zeocin. Hundreds of transformants were obtained by one transformation using the linearized plasmid.

  In order to test recombinant yeast clones for QC expression, recombinants were cultured for 24 hours in 10 ml conical tubes containing 2 ml BMGY. The yeast was then centrifuged and resuspended in 2 ml BMMY containing 0.5% methanol. This concentration was maintained up to 72 hours by adding methanol every 24 hours. Thereafter, the QC activity in the supernatant was determined. The presence of the fusion protein was confirmed by Western blot analysis using an antibody directed against a 6 × histidine tag (Qiagen). Clones that showed the highest QC activity were selected for further experiments and fermentation.

(Large scale expression in fermenter)
QC expression was performed in a 5 l reactor (Biostat B, B. Braun biotech), essentially as described in “Pichia Fermentation Process Guidelines” (Invitrogen). Briefly, cells were cultured in a fermentation basal salt medium supplemented with trace salts and glycerol as the sole carbon source (pH 5.5). Large amounts of cells accumulated during the initial batch phase of about 24 hours and the subsequent feed batch phase of about 5 hours. Once 200 g / l wet cell weight was achieved, methanol was used to induce QC expression by applying a three-step feed profile during the entire fermentation time of approximately 60 hours. Thereafter, the cells were removed from the supernatant containing QC by centrifugation at 6000 × g and 4 ° C. for 15 minutes. The pH was adjusted to 6.8 by addition of NaOH and the resulting cloudy solution was centrifuged again at 37000 × g, 4 ° C. for 40 minutes. If turbidity persisted, a further filtration step was applied using a cellulose membrane (pore width 0.45 μm).

(Purification of 6x histidine-tagged QC expressed in P. pastoris)
His-tagged QC was purified by first immobilized metal affinity chromatography (IMAC). In a typical purification, 1000 ml of the culture supernatant is applied to a 5 ml / ml Chelating Sepharose FF column (1.6 x 20 cm, Pharmacia) packed with Ni ²⁺ equilibrated with 50 mM phosphate buffer pH 6.8 containing 750 mM NaCl. Applied at a flow rate of minutes. After washing with 10 column volumes of equilibration buffer and equilibration buffer containing 5 column volumes of 5 mM histidine, the bound protein is exchanged for 50 mM phosphate buffer, pH 6.8 containing 150 mM NaCl and 100 mM histidine. Elute. The resulting eluate was dialyzed overnight at 4 ° C. against 20 mM Bi-Tris / HCl (pH 6.8). The QC was then further purified by anion exchange chromatography Mono Q6 column (BioRad) equilibrated with dialysis buffer. The fraction containing QC was loaded onto the column using a flow rate of 4 ml / min. The column was then washed with equilibration buffer containing 100 mM NaCl. Elution was performed with two gradients that yielded an equilibration buffer containing 30 or 5 column volumes of 240 mM and 360 mM NaCl, respectively. Six ml fractions were collected and analyzed for purity by SDS-PAGE. Fractions containing homogeneous QC were pooled by ultrafiltration and concentrated. Glycerol was added to a final concentration of 50% for long-term storage (-20 ° C). Protein was quantified according to the method of Bradford or Gill and von Hippel (Bradford, MM 1976 Anal Biochem 72, 248-254; Gill, SC and von Hippel, PH 1989 Anal Biochem 182, 319-326).

(Expression and purification of QC in E. coli)
The construct encoding QC was transformed into M15 cells (Qiagen) and selective LB agar plates were cultured at 37 ° C. Protein expression was performed at room temperature in LB medium containing 1% glucose and 1% ethanol. Expression was induced overnight with 0.1 mM IPTG when the culture reached an OD ₆₀₀ of approximately 0.8. After one cycle of freeze-thawing, the cells were lysed for approximately 30 minutes at 4 ° C. by addition of 2.5 mg / ml lysozyme in 50 mM phosphate buffer, pH 8.0 containing 300 mM NaCl and 2 mM histidine. The solution is clarified by centrifugation at 37000 × g for 30 minutes at 4 ° C., followed by filtration applying a glass frit (DNA separation), and two times applying a cellulose filter against the crude and fine precipitates. A further filtration step was performed. The supernatant (about 500 ml) was applied to a Ni ²⁺ -affinity column (1.6 × 20 cm) at a flow rate of 1 ml / min. QC elution was performed with 50 mM phosphate buffer containing 150 mM NaCl and 100 mM histidine. The fraction containing QC was concentrated by ultrafiltration.

(Reference Example 2: MALDI-TOF mass spectrometry)
Matrix-assisted laser desorption / ionization mass spectrometry was performed by using a Voyager De-Pro (Applied Biosystems, Darmstadt) equipped with a linear time-of-flight analyzer. The instrument was equipped with a 337nm nitrogen laser, a potential acceleration source, and a 1.4m flight tube. The detector operation was in positive ion mode. Sample (5 μl) was mixed with the same volume of matrix solution. For matrix solutions, we prepared sinapinic acid prepared by dissolving 20 mg sinapinic acid (Sigma-Aldrich) /0.1% TFA in water (1/1, v / v) in 1 ml acetonitrile. used. A small volume (≦ 1 μl) of the matrix-analyte-mixture was transferred to the probe tip.

For long-term testing of Glu ¹ -cyclization, Aβ-derived peptides were incubated at 30 ° C. in 100 μl 0.1 M sodium acetate buffer, pH 5.2, or 0.1 M Bis-Tris buffer pH 6.5. Peptides were applied at a concentration of 0.5 mM [Aβ3-11a] or 0.15 mM [Aβ3-21a] and 0.2 U QC was added all for 24 hours. In the case of Aβ3-21a, the assay included 1% DMSO. At different time points, samples are removed from the assay tubes, peptides are extracted using ZipTips (Millipore) according to manufacturer's recommendations, mixed with matrix solution (1: 1 v / v) and then mass spectra Recorded. Negative controls included neither QC nor heat-inactivated enzyme. For inhibitor studies, the sample composition was the same as above except for added inhibitory compounds (5 mM benzimidazole or 2 mM 1,10-phenanthroline).

(Example 1: Preparation and expression of human MCP-1 in mammalian cell culture)
Cell lines and media Human neuroblastoma cell line SH-SY5Y, human embryonic kidney cell line HEK293, and human monocyte cell line THP-1 in an appropriate cell medium (DMEM, SH-SY5Y, and HEK293) 10% FBS for (RPMI1640, 10% FBS for THP-1), 5% CO ₂ (HEK293, THP-1), or 10% CO ₂ (SH-SY5Y) The culture was performed at 37 ° C. in an atmosphere.

Isolation of human MCP-1 Full length cDNA of human MCP-1 was isolated from SH-SY5Y cells using RT-PCR. Total RNA of SH-SY5Y cells was reverse transcribed with SuperScript II (Invitrogen), followed by human MCP-1, primers hMCP-1-1 (sense), and hMCP-1-2 (antisense) (Table 1) Was amplified with Pfu-DNA-Polymerase (Promega) at a 1: 12,5 dilution of the cDNA product produced in a 25 μl reaction. The resulting PCR product was cloned into the vector pcDNA 3.1 using HindIII and NotI restriction sites and sequences and confirmed by DNA sequencing.

Site-directed mutagenesis of human MCP-1 Detection of the first (ΔQ1) and first and second (ΔQ1P2) amino acids of mature human MCP-1 was performed using primer ΔQ1-1 for ΔQ1 (Table 1). And ΔQ1-2, and ΔQ1P2 (Table 1), and primers ΔQ1P2-1 and ΔQ1P2-2 were used to produce by site-directed mutagenesis. Parental DNA was digested with DpnI. The pcDNA 3.1 plasmid carrying the mature human MCP-1 deletion ΔQ1 and ΔQ1P2 was transformed into E. coli JM109. Ampicillin resistant clones were confirmed by sequencing and then isolated for cell culture purposes using the EndoFree Maxi Kit (Qiagen).

Expression of human MCP-1 N-terminal mutant in HEK293 cells For expression of human MCP-1 N-terminal mutant, HEK293 cells were cultured in collagen I-coated 6-well dishes and 80% confluent And transfected using Lipofectamin 2000 (Invitrogen) according to the manufacturer's manual and incubated in the transfection solution for 5 hours. Cells were then collected overnight in normal growth medium. The next day, the cells were further incubated in growth medium for 24 hours. For analysis of the efficacy of QC inhibition, cells were incubated for 24 hours in the absence or presence of specific inhibitors. After 24 hours, media containing human MCP-1 mutants were collected and investigated for chemotaxis performance by migration assay. Furthermore, an aliquot of cell culture supernatant was stored at -80 ° C. for quantification of human MCP-1 concentration using human MCP-1-ELISA (Pierce).

Transwell chemotaxis assay The chemotaxis assay was performed using 24-well TransWell plates (Corning) with a pore size of 5 μm. A medium containing the human MCP-1 mutant expressed in HEK293 was used as a chemoattractant. To help this, 600 μl of N-terminal human MCP-1 variant medium was applied to the lower chamber of the TransWell plate at undiluted or 1: 3, 1:10, and 1:30 dilutions of RPMI1640. Furthermore, undiluted medium of HEK293 cells transfected with the vector control was applied to the lower chamber as a negative control. THP-1 cells were collected, resuspended in RPMI 1640 at a concentration of 1 * 10 ⁶ cells / 100 μl and applied to the upper chamber in 100 μl aliquots. Cells were moved towards chemoattractant for 2 hours at 37 ° C. Thereafter, cells from the upper chamber were discarded and the lower chamber was mixed with 50 μl of 70 mM EDTA in PBS and incubated at 37 ° C. for 15 minutes to release the cells attached to the membrane. The cells that migrated to the lower chamber were then counted using a cell counter system (Schrfe System). The chemotaxis index was calculated by dividing the cells that migrated to the stimulus by the cells that migrated to the negative control.

(Example 2: Study on proteolysis of human MCP-1 _(1-76) )
Method N-terminal degradation by recombinant human DP4
Full length recombinant human MCP-1 _(1-76) encoded by the nucleic acid sequence as shown in SEQ ID NO: 2 obtained in Example 1 above, starting with N-terminal glutamine (Peprotech) : 1) was dissolved in 25 mM Tris / HCl pH 7.6 at a concentration of 10 μg / ml. The MCP-1 solution was recombinant human QC (0.0006 mg / ml) (obtained according to Reference Example 1 above, SEQ ID NO: 3 for the nucleic acid sequence and SEQ ID NO: 4 for the amino acid sequence) and 3 at 30 ° C. Pre-incubated for a period of time followed by incubation with recombinant human DP4 (0.0012 mg / ml) at 30 ° C. (see FIG. 1) or with DP4 without prior QC application. The resulting DP4 cleavage product was analyzed after 0 min, 15 min, 30 min, 1 h, 4 h, and 24 h using Maldi-TOF mass spectrometry.

N-terminal degradation by human rheumatoid synovial fibroblast MMP-1 Human recombinant MCP-1 with N-terminal glutaminyl instead of pyroglutamyl residue (Peprotech) at a concentration of 10 mM / ml, 25 mM Tris / HCl (pH 7.6) ). Activated MMP-1 enzyme precursor (Calbiochem) from human rheumatoid synovial fibroblasts using 25 mM p-aminophenylmercury acetate (APMA), 37 ° C. in 10: 1 APMA: enzyme mixture For 3 hours in 0.1N NaOH. MCP-1 was preincubated with recombinant human QC (0.0006mg / ml) at 30 ° C for 3 hours, followed by incubation with MMP-1 at 30 ° C or without prior QC application Incubated with MMP-1. The resulting MMP-1 cleavage products were analyzed after 0 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 24 hours using Maldi-TOF mass spectrometry.

N-terminal degradation by human rheumatoid synovial fibroblast MMP-1 and recombinant human DP4
Human recombinant MCP-1 (Peprotech) starting with N-terminal glutamine was dissolved in 25 mM Tris / HCl (pH 7.6) at a concentration of 10 μg / ml. MMP-1 enzyme precursor (Calbiochem) from human rheumatoid synovial fibroblasts was activated using 25 mM p-aminophenylmercury acetate (APMA) dissolved in 0.1 N NaOH. The 10: 1 APMA: enzyme mixture was incubated at 37 ° C. for 3 hours. MCP-1 solution should be preincubated with recombinant human QC (0.0006mg / ml) at 30 ° C for 3 hours and then incubated with MMP-1 and DP4 at 30 ° C, or QC applied Without incubation with MMP-1 and DP4. The resulting MMP-1 cleavage products were analyzed after 0 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 24 hours using Maldi-TOF mass spectrometry.

(Example 3: 1- (3- (1H-imidazol-1-yl) propyl) -3 hydrochloride, a QC-specific inhibitor for the promotion of cuff-induced atherosclerosis in ApoE3 * Leiden mice Effect of-(3,4-dimethoxyphenyl) thiourea (hereinafter also named QCI) Timeline
Thirty male ApoE3 * Leiden mice (12 weeks of age) received a light hypercholesterolemic diet for 3 weeks prior to surgical cuff placement.

  Three weeks later, the mice received surgical non-shrink cuff placement (day 0) and were divided into two groups matched for plasma cholesterol levels. Mice were either control (acidified) drinking water or 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride, which is a QC specific inhibitor Any of the drinking water containing was received at a concentration of 2.4 mg / ml. Seven days after the start of treatment, the inhibitor concentration was reduced to 1.2 mg / ml. Atherosclerosis, 5 mice in each group were sacrificed 2 days later for monocyte adhesion and invasion analysis, and 10 mice were sacrificed 2 weeks later for histomorphometric analysis The promotion of type lesions and the inhibition of neointimal formation were quantified.

Cuff placement surgical procedure During surgery, mice were anesthetized with an intraperitoneal injection of 5 mg / kg Dormicum, 0.5 mg / kg Domitor, and 0.05 mg / kg fentanyl. The cocktail can be fully anesthetized for at least 1 hour and rapidly antagonized with Antisedan 2.5 mg / kg and Annexe 0.5 mg / kg.

  A 1 cm incision is made inside the leg and the femoral artery is dissected 3 mm from the femoral nerve and femoral vein. The femoral artery is circularized by ligation, a non-shrinkable microporous polyethylene tube (0.4 mm inner diameter, 0.8 mm outer diameter, 2 mm length) is opened longitudinally, and the femoral artery Attach a sleeve loosely around. The cuff closes up with two ligature knots. The skin is closed with a continuous suture.

  After surgery, the animals were antagonized for several hours and placed in a clean cage on a heating pad.

Animal sacrifice For histological analysis, animals were sacrificed 2 or 14 days after cuff placement. After anesthesia, the chest was opened and mild pressure-perfusion (100 mmHg) with 4% formaldehyde was performed by cardiac puncture for 3 minutes. After perfusion, a 2 cm longitudinal incision was made on the inside of the leg and the cuffed femoral artery was collected as a whole, fixed in 4% formaldehyde overnight and processed into process paraffin.

Monocyte adhesion and analysis of MCP-1 expression Adherence of common leukocytes to the activated endothelium of the cuffed vascular wall, and in particular the monocyte / macrophage cuff, is a cross-sectional microscope collected 2 days after cuff placement Analyzed by analysis. Count the number of common white blood cells identified as cells that adhere and / or infiltrate, adhere to the luminal side of the blood vessel segment, and in particular the number of monocytes / macrophages, as cells per cross section or as a cross section Shown as per defined area. Monocytes were identified by specific immunohistochemical staining with a polyclonal rabbit AIA31240 antibody that recognizes monocytes and macrophages. In addition to these sections, immunohistochemical staining specific for MCP-1 was performed.

Analysis of vascular remodeling and accelerated atherosclerosis Defect wall remodeling, accelerated atherosclerosis, and neointimal formation were analyzed morphometrically in all mice sacrificed after 14 days did. A complete comparison between the two groups was performed for all relevant vessel wall parameters (neointimal formation, vessel periphery (ie external remodeling), media thickness, lumen stenosis). The promotion of atherosclerosis was analyzed by immunohistochemical staining for macrophages and foam cells in the lesion area with AIA31240 antibody. Furthermore, these sections were also stained for MCP-1.

Example 4: Proteolysis of human MCP-1 _(1-76) by dipeptidyl-peptidase 4 (DP4), aminopeptidase P, and by proteases present in human serum
N-terminal degradation with recombinant human aminopeptidase P Human recombinant MCP-1 with N-terminal glutaminyl instead of pyroglutamyl residue (Peprotech) was dissolved in 25 mM Tris / HCl, pH 7.6 at a concentration of 10 μg / ml. MCP-1 was incubated at 30 ° C. with 30 μg / ml aminopeptidase P (R & D Systems). Gln ¹ -MCP-1 was used without pGlu-modification or preincubated with recombinant human QC (6 μg / ml) at 30 ° C. for 3 hours to produce pGlu. The resulting aminopeptidase P cleavage product was analyzed after 0 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 24 h using Maldi-TOF mass spectrometry.

N-terminal degradation of MCP-1 by recombinant human DP4 in the absence and presence of QC-specific inhibitors
Recombinant human MCP-1 _(1-76) encoded by the nucleic acid sequence as shown in SEQ ID NO: 2 obtained in Example 1 above, starting from N-terminal glutamine (Peprotech) 1) was dissolved in 25 mM Tris / HCl pH 7.6 at a concentration of 10 μg / ml. The MCP-1 solution was preincubated with recombinant human QC (0.0006 mg / ml) (obtained according to Reference Example 1 above) at 30 ° C. for 3 hours, followed by recombinant human DP4 (0.0012 mg / ml). ) At 30 ° C. (see FIG. 1) or incubated with DP4 without prior QC application. In addition, incubation of recombinant human QC and Gln ¹ -MCP-1 was performed with 10 μM 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride. Carried out in the presence of The resulting DP4 cleavage product was analyzed after 0 min, 15 min, 30 min, 1 h, 2 h, and 4 h using Maldi-TOF mass spectrometry.

N-terminal degradation of human MCP-1 in human serum Human recombinant MCP-1 (Peprotech) with N-terminal glutaminyl instead of pyroglutamyl residue was dissolved in 25 mM Tris / HCl (pH 7.6) at a concentration of 100 μg / ml . MCP-1 can be preincubated with recombinant human QC (0.006mg / ml) at 30 ° C for 3 hours, followed by incubation with human serum at 30 ° C, or human serum without addition of QC. Incubated. Cleavage products were obtained for Gln ¹ -MCP-1 using Maldi-TOF mass spectrometry for 0 min, 10 min, 30 min, 1 h, 2 h, 3 h 5 h, and 7 h, and pGlu ¹ -MCP -1 was analyzed after 0 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 7 hours, and 24 hours.

(Example 5: Degradation of human MCP-2, MCP-3, and MCP-4)
N-terminal degradation of human MCP-2 by DP4 Human recombinant MCP-2 (Peprotech) having N-terminal glutaminyl instead of pyroglutamyl residue was dissolved in 25 mM Tris / HCl (pH 7.6) at a concentration of 10 μg / ml. MCP-2 can be preincubated with recombinant human QC (0.0006 mg / ml) at 30 ° C for 3 hours, followed by incubation with recombinant human DP4 (0.0012 mg / ml) at 30 ° C, or QC And incubated with recombinant human DP4 (0.0012 mg / ml) without preincubation. The resulting DP4 cleavage products were analyzed after 0 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 24 h using Maldi-TOF mass spectrometry.

N-terminal degradation of human MCP-3 by DP4 Human recombinant MCP-3 (Peprotech) with N-terminal glutaminyl instead of pyroglutamyl residue was dissolved in 25 mM Tris / HCl (pH 7.6) at a concentration of 10 μg / ml. MCP-3 can be preincubated with recombinant human QC (0.0006 mg / ml) at 30 ° C. for 3 hours followed by incubation with recombinant human DP4 (0.00012 mg / ml) at 30 ° C. Incubated with recombinant human DP4 (0.00012 mg / ml) without QC application. The resulting DP4 cleavage products were analyzed after 0 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 24 h using Maldi-TOF mass spectrometry.

N-terminal degradation of human MCP-4 by DP4 Human recombinant MCP-4 (Peprotech) with N-terminal glutaminyl instead of pyroglutamyl residue was dissolved in 25 mM Tris / HCl (pH 7.6) at a concentration of 10 μg / ml. MCP-4 can be preincubated with recombinant human QC (0.0006 mg / ml) at 30 ° C. for 3 hours followed by incubation with recombinant human DP4 (0.00006 mg / ml) at 30 ° C. Incubated with recombinant human DP4 (0.00006 mg / ml) without QC application. The resulting DP4 cleavage products were analyzed after 0 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 24 h using Maldi-TOF mass spectrometry.

(Example 6: chemotaxis performance of different N-terminal mutants of human MCP-1, MCP-2, MCP-3, MCP-4)
Chemotactic performance of N-terminal mutants of human MCP-1 MCP-1 starting with glutamine 1 (Gln ¹ -MCP-1) (Peprotech) (i) Recombinant human to produce pGlu ¹ -MCP-1 QC, (ii) human recombinant DP4 to produce Asp ³ -MCP-1, (iii) human synovial fibroblast MMP-1 to produce Ile ⁵ -MCP-1, and Pro ² -MCP Incubated with human recombinant aminopeptidase P to produce -1. Produced MCP-1 variants at concentrations of 1, 5, 10, 50, 100, 500, and 1000 ng / ml were tested using the THP-1 chemotaxis assay (n = 3).

Human MCP-1 chemotaxis performance in the absence or presence of QC inhibitors
MCP-1 with N-terminal glutamine (Gln ¹ -MCP-1) (Peprotech) is combined with recombinant human QC and DP4 (Gln ¹ -MCP-1 + QC + DP4) and human recombinant DP4 alone (Gln ¹ -MCP + DP4), and 10 μM QC inhibitor 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea, and DP4 Incubated with recombinant human QC (Gln ¹ -MCP-1 + QC + QCI + DP4). Produced MCP-1 variants at concentrations of 1, 5, 10, 50, 100, 500, and 1000 ng / ml were tested using a chemotaxis assay (n = 3).

Comparison of chemotaxis performance of variants of human MCP-1, MCP-2, MCP-3, and MCP-4 with N-terminal glutaminyl or pyroglutamyl residues
N-terminal glutamine (Peprotech), or human MCP-1, MCP-2, MCP-3, and MCP-4 with pyroglutamyl-residue (1: 100 dilution of human recombinant QC and Gln ¹ -MCP) Incubation at 30 ° C for 2 hours) was tested for chemotaxis performance. Specific MCPs at concentrations of 1, 5, 10, 50, 100, 500, and 1000 ng / ml were tested using a chemotaxis assay (n = 3).

Comparison of the chemotaxis performance of mutants of human MCP-1, MCP-2, MCP-3, and MCP-4 with N-terminal glutaminyl residues with their respective DP4 cleavage products
Applying human MCP-1, MCP-2, MCP-3, and MCP-4 starting with N-terminal glutamine (Peprotech) directly to the chemotaxis assay, MCP-1, MCP-2, MCP-3, and It was compared with the chemotaxis performance of the DP4 cleavage product of MCP-4. For production of DP4 cleavage products, each MCP was incubated with human recombinant DP4 at a 1: 100 dilution for 2 hours at 30 ° C. prior to assay. Specific MCPs at concentrations of 1, 5, 10, 50, 100, 500, and 1000 ng / ml were tested using a chemotaxis assay (n = 3).

Example 7: Application of a QC inhibitor to a model of LPS-induced sepsis in rats
Preparation
The QC inhibitor 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride is 0.9% (w / v) saline. Lower doses were obtained by serial dilution using 0.9% (w / v) saline. In addition, a stock solution of LPS (1 mg / mL) is prepared using 0.9% (w / v) saline and diluted using 0.9% (w / v) saline. Provided the concentration required for dosing.

Concentration Dose levels are expressed in terms of the amount of inhibitor administered, regardless of purity or active content.

Species male Han Wistar rats were obtained from Charles River (UK) Ltd., Margate, Kent.

Acclimatization and health procedures Upon arrival, all animals were examined for unhealth. Animals were acclimated for a period of at least 5 days prior to dosing. During this time, animals were identified by their cage label. Veterinary examinations were performed before the start of any experimental procedure to confirm their suitability for the study.

Experimental design The study was conducted over 2 days (5 animals from each treatment group on each day). Food and water were available as appropriate except when animals were removed from the home cage for study procedures. Each animal uses a specific dose volume of 2 mL / kg as a slow bolus dose, at low, medium and high doses (Table 2), 3.5 hours and 0.5 hours before LPS administration, We received two single intravenous doses of vehicle or QC inhibitor 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea. Thirty minutes after the last administration of vehicle or test article, each animal received an intraperitoneal injection of LPS or saline using a specific dose volume of 5 mL / kg. Individual dose volumes were based on individual body weights obtained on the day of dosing. The treatment groups used for the study are shown in Table 2.

Sampling and TNFα determination Peripheral blood samples were collected 2 hours after LPS. Blood samples were centrifuged at 2300 × g for 10 minutes at 4 ° C. and then analyzed for TNFα. Samples were analyzed using a quantitative sandwich enzyme immunoassay.

Example 8: Evaluation of QC inhibitors in a mouse model of thioglycolate-induced peritonitis
Animals For each experiment, C57 / Bl6J wild type mice were purchased from Charles River Laboratories Inc. For each experiment, mice were matched in age and sex.

Induction of peritonitis induced by thioglycolate For the induction of peritonitis, mice were injected intraperitoneally (ip) with 25 ml / kg body weight of sterile 8% (w / v) thioglycolate (Sigma) -Aldrich; Time: t = 0). At different time points before and after thioglycolate application, mice were injected ip with various concentrations of QC inhibitors. For peritoneal lavage, animals were anesthetized using 2% isoflurane. Peritoneal exudates were collected at time points (4, 24 hours) after thioglycolate injection by washing the peritoneum with 8 ml of sterile phosphate buffered saline (PBS). The wash was then centrifuged to pellet the cells and stained for FACS analysis.

Analysis of cellular composition of collected exudates using FACS analysis Samples were stained for BD Trucount tubes according to manufacturer's instructions (BD Trucount tubes; catalog number 340334; BD Biosciences). Cells were blocked with CD16 / 32 (Caltag) and stained with the following antibodies for 15 minutes: CD3-FITC (Caltag) / CD13-PE (BD) / F4 / 80-APC (Caltag); Moma2-FITC (Acris ), And IgG1-PE (BD) / IgG2a-APC (Caltag) as an isotype control. After staining, the cells were lysed with BD FACSLyse (BD) for 15 minutes at room temperature in the dark. As a reference standard, flow cytometric analysis of 5000 beads per sample was performed on a BD FACSCalibur (BD Biosciences).

Results Preparation and expression of human MCP-1 in mammalian cell culture Amplification of human MCP-1 from human neuroblastoma cell line SH-SY5Y RNA resulted in a 300 bp PCR product. Sequencing of the isolated cDNA revealed an unaltered single nucleotide polymorphism of codon 105 encoding cysteine 35.

  Expression of the human MCP-1 mutant in HEK293 reaches high levels in the cell culture supernatant as monitored by human MCP-1 ELISA. According to this, expression of MCP-1 (WT) and MCP-1 (ΔQ1) (FIG. 5C) and 10 μM hydrochloric acid 1- (3- (1H-imidazol-1-yl) propyl) -3- (3, The level between MCP-1 (WT) in the absence or presence of 4-dimethoxyphenyl) thiourea (FIG. 7A) does not change significantly. However, the expression of MCP-1 (ΔQ1P2) is reduced by 28% compared to MCP-1 (WT). The supernatant was collected and applied to the TransWell migration assay (see FIGS. 4, and 5C and D for this point).

TransWell chemotaxis assay Purified human MCP-1 exhibits a bell-shaped chemotaxis dose response curve when attracted, for example, monocytes are optimal at about 1-50 ng / ml. Therefore, to achieve the optimal working concentration of MCP-1 for the chemotaxis assay that attracts THP-1 monocytes, the cell culture supernatant containing the produced MCP 1 mutant was serially diluted.

  After expression of MCP-1 (WT) and MCP-1 (ΔQ1), the concentration of MCP-1 mutant was not significantly different (FIG. 5C). Application of MCP-1 (WT) to the chemotaxis assay caused a chemotactic response in THP-1 cells (FIG. 5D), indicated by a high chemotaxis index. However, MCP-1 (ΔQ1) was unable to induce chemotaxis of THP-1 and was suggested by a chemotaxis index of approximately 1 (FIG. 5D). These results support the previous results that N-cleaved MCP-1 is inactive. This finding is further evidenced by the inability of MCP-1 (ΔQ1P2) to induce chemotaxis of THP-1 cells (FIG. 6B). Expression of MCP-1 (WT) in HEK293 cells has no effect on the absence or presence of chemotactic cytokines (chemokines). However, application of chemokines causes significantly lower chemotaxis of THP 1 cells at 1: 3 and at 1:10 dilution (FIG. 7B). This is because N of MCP-1 (WT) by 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride, a QC-specific inhibitor This suggests inactivation of MCP-1 (WT) by blocking terminal pGlu- formation and thus by N-terminal proteolytic degradation or by only blocking pGlu formation.

Study on proteolytic degradation of human MCP-1 _{(1-76) In} circulation, MCP-1 is protected by an N-terminal pGlu-residue that confers resistance to N-terminal cleavage by aminopeptidases such as DP4 Is done. As a result of QC inhibitor administration, the unprotected N-terminal group is easily cleaved by DP4. N-terminal truncation then causes inactivation of human MCP-1 (FIGS. 5 and 6). MMP-1 inactivates mature MCP-1 by cleavage of the four N-terminal amino acids (pE / QPDA). The reaction is independent of the presence of the N-terminal pGlu residue. This process reflects the situation of MCP-1 inactivation in the circulation. The resulting cleavage product MCP 1 _(5-76) has been shown to be present in plasma and resembles a naturally occurring CCR2 receptor antagonist. This experiment shows the finding that MMP-1 cleavage is slightly faster with the N-terminal glutamine residue (FIG. 2A: 2 hours, 4 hours vs. 2B: 2 hours, 4 hours). Furthermore, incubation of human MCP-1 with an N-terminal Gln residue (FIG. 3A) with human DP4 and human MMP-1 shows enhanced degradation compared to pGlu-MCP-1 (FIG. 3B). .

  Taken together, the results indicate that N-terminal pGlu formation represents a protective mechanism, as implied by N-terminal degradation by post-proline cleavage enzymes such as DP4, aminopeptidase, and by results with MMP-1. It implies that it is also resistant to endoproteases to a certain extent. Blocking N-terminal pGlu formation by application of a QC inhibitor causes more rapid inactivation of human MCP-1.

Analysis of vascular remodeling and promotion of atherosclerosis in ApoE3 * Leiden mice
Treatment of cuff-induced promotion of atherosclerosis in ApoE3 * Leiden mice has no effect on the total area within the outer diameter of the vascular segment (Figure 8A) and mild in the remaining lumen Although an increase can be observed, there was no statistically significant effect on the remaining lumens (FIG. 8B). However, 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride is a sufficient 40% reduction in the percentage of lumen constriction (Fig. 9A) and a 45% reduction in the area of neointima formation (FIG. 9B). Both values are statistically significant. Furthermore, the inhibitor decreased the media area (Figure 10A) and the intima / media ratio (Figure 10B), but the decrease in the intima / media ratio was statistically significant. (P <0.102).

  Analysis of cellular composition in specific vascular wall layers shows no difference in the relative contribution of smooth muscle cells and macrophages / foam cells to both media and pericardial composition after 2 and 14 days ( Figure 15). Monocytes in the vessel wall due to the effect of 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea on MCP-1 and thus on monocyte attraction A more specific effect on macrophage content can be expected, but as recently discovered and published by Schepers, A. 2006 Arterioscler Thromb Vasc Biol. 26, 2063-2069, MCP-1 It should also be noted that it has a direct effect on smooth muscle cell proliferation.

Analysis of monocyte adhesion and MCP-1 expression of mild hypercholesterolemia with 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride Treatment of ApoE3 * Leiden mice (plasma cholesterol level 12-15 mM) resulted in a sufficient reduction of 45% total adherent cells after 2 days (p <0.05). Specific analysis of adhering monocytes revealed an even more potent decrease of 67% (p <0.05) on treated cuffed vessel segments (FIG. 11).

MCP-1 expression is 1- (3- (1H-imidazol-1-yl) propyl hydrochloride) -3- (3, 2 days after surgery, which is the moment of highest rise in MCP-1 expression in the model used There was a decrease in the vascular segment of mice treated with 4-dimethoxyphenyl) thiourea (Figure 12, 13A, 14A). These results indicate that 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride is administered early after vascular injury in the lesion. , Shows that a decrease in MCP-1 expression can be detected in both the media of the vascular wall segment and the intima (ie, inside the inner elastic lamina). Analysis of the relative area of the cross section positive for MCP-1 reduced 52% (P = 0.01) of MCP 1 expression in the media and 36% (P = 0.001) in the intima (Figure 14A) Became clear. Analysis of the absolute area positive for MCP-1 (expressed as μm ² positive per cross section) in the media (41% reduction, p = 0.09) and intima (40% reduction, p = 0.05) A similar decrease in MCP-1 expression is evident, but the decrease in the media is not statistically significant (Student T test) (FIG. 13A).

  At the 14 day time point, overall MCP-1 expression was lower than that observed for the initial time point, as opposed to MCP-1 when neointimal formation / atherosclerosis promotion proceeded Decreased expression cannot be monitored in the media, or in neointimal (FIGS. 13B, 14B), and 1- (3- (1H-imidazole hydrochloride) only for the time of strong induction of MCP-1. Suggests the effect of -1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea.

  Taken together, these data indicate that oral administration of 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride is in the ApoE3 * Leiden cuff model Shows beneficial effects on vascular remodeling after intervention and promotion of atherosclerosis.

Proteolysis of human MCP-1 _(1-76) by human aminopeptidase combined with QC-specific inhibitors and human serum
QC inhibitor 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4, a QC inhibitor for the production of N-terminal pGlu-residue and its subsequent effect on proteolytic stability To further illustrate the effect of -dimethoxyphenyl) thiourea, human MCP-1 with N-terminal glutamine (FIG. 17A) or pyroglutamic acid (FIG. 17B) was incubated with DP4. N-terminal pGlu- formation was achieved by pre-incubation of human QC and precursor, reflecting a physiological maturation process. As expected, in the absence of preincubation with human QC, MCP-1 is sensitive to DP4 cleavage (FIG. 17A). In contrast, preincubation with human QC causes the formation of an N-terminal pGlu-residue and thus its protection against DP4 cleavage (FIG. 17B). In addition, human MCP-1 with human QC in the presence of the QC inhibitor 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride Pre-incubation results in inhibition of QC and thus prevention of pGlu-MCP-1 formation. Inhibition of pGlu-MCP-1 formation by 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride prevents the MCP-1 peptide from undergoing DP4 cleavage To make it sensitive again (Fig. 17C). Thus, inhibition of QC causes destabilization of the N-terminus of MCP-1 in vitro and in vivo.

Similar to the N-terminal cleavage of human MCP-1 by DP4, incubation of recombinant human aminopeptidase P with Gln ¹ -MCP-1 causes an unprotected N-terminal cleavage. As a result, aminopeptidase P cleaves the N-terminal amino acid between Gln ¹ and Pro ² to release the N-terminal glutaminyl residue (FIG. 16A). However, preincubation of Gln ¹ -MCP-1 with human QC results in the formation of an N-terminal pGlu-residue, thus resulting in protection against aminopeptidase P cleavage (FIG. 16B). Thus, the formation of the N-terminal pGlu-residue is also a protective mechanism against aminopeptidase P cleavage and possibly against the cleavage of all other proline-specific aminopeptidases. For further studies on the proteolytic stability of human MCP-1, the data obtained by incubation of purified protease with MCP-1 was verified by incubation of human serum with human MCP-1. Incubation of human serum with human Gln ¹ -MCP-1 shows N-terminal truncation of the substrate and release of the ^first two amino acids (Gln ¹ Pro ² ). In addition, QC activity in plasma competes with N-terminal proteolysis, stabilizes MCP-1, and approximately 60% of cleaved Asp ³ -MCP-1 and 40% of full-length pGlu ¹ -MCP-1 End with the final ratio (Figure 18A). Furthermore, preincubation of human QC with human MCP-1 leads to the formation of N-terminal pGlu-residues and thus stabilization of human MCP-1. No degradation of pGlu ¹ -MCP-1 was observed at least for the selected time frame and serum dilutions (FIG. 18B). In addition, incubation of MCP-1 in serum in the presence of the 9.6 μM DP4-inhibitor isoleucyl-Thiyzolidide also prevented N-terminal degradation, which means that MCP-1 is DP4 or DP4 in human serum It is proved that it is degraded by a similar activity (Figure 18C).

Proteolysis of human MCP-2, MCP-3, and MCP-4 Similar to the N-terminal degradation of human MCP-1, other human MCPs for N-terminal cleavage by DP4, namely MCP-2, MCP-3, and The sensitivity of MCP-4 was investigated. As previously observed for MCP-1, the N-terminal pGlu-residue is driven by DP4 to MCP-2 (Figure 19B), MCP-3 (Figure 20B), and MCP-4 (Figure 21B). Protect from proteolysis. However, uncyclized mutants starting with N-terminal glutamine are Gln ¹ -MCP-2 (FIG. 19A), Gln ¹ -MCP-3 (FIG. 20A), and Gln ¹ -MCP-4 (FIG. 19). As shown for 21 A), it is easily cleaved by DP4. Thus, the N-terminal pGlu-residue stabilizes all MCPs against cleavage by aminopeptidases such as DP4. Thus, the proposed concept for reducing QC activity in vivo to promote turnover and chemotaxis and receptor activation applies to all members of the MCP-family.

Chemotaxis performance of different N-terminal mutants of human MCP-1, MCP-2, MCP-3, MCP-4 Investigating the effect of different N-terminal mutants of MCP-1 on the ability to attract human THP-1 monocytes Gln ¹ -MCP-1, pGlu ¹ -MCP-1, aminopeptidase P cleavage product Pro ² -MCP-1, DP4 cleavage product Asp ³ -MCP-1, and MMP-1 cleavage product Ile ⁵ -MCP -1 was tested in an in vitro chemotaxis assay. Full-length MCP-1 with an N-terminal glutaminyl or pyroglutamyl-residue may be equally potent to attract THP-1 monocytes with a maximum response between 50 ng / ml and 100 ng / ml I found it. In contrast, cleavage of MCP-1 by aminopeptidase P (Pro ² -MCP-1) and DP4 (Asp ³ -MCP-1) causes a loss of the ability of the respective mutants. Changing the dose response curve to a higher concentration requires eliciting a maximal response, which corresponds to inactivation of MCP-1 by N-terminal truncation. MMP-1 cleavage product (Ile ⁵ -MCP-1) has a maximum equivalent to Glu ¹ -MCP-1 and pGlu ¹ -MCP-1 between 50 ng / ml and 100 ng / ml, but full length MCP Compared to -1, the amount of cells migrating to this mutant (ie, chemotaxis performance) is very low (Figure 22).

To further investigate the role of QC in stabilizing MCP-1 and its effect on THP-1 monocyte migration, Gln ¹ -MCP-1 was incubated with human DP4. In parallel samples, MCP-1 was preincubated with human QC prior to DP4 application. As expected, the resulting dose response curve implies the proteolytic stability of pGlu ¹ -MCP-1 as reflected by the maximal response at 50-100 ng / ml. In contrast, in the absence of QC, Gln ¹ -MCP-1 is cleaved by DP4, which results in a higher MCP-1 concentration (500-1000 ng / ml) required to elicit a maximal response Causes a change in the dose-response curve. In addition, QC and the QC inhibitor 1- (HCl, as observed by the change in dose response curve to higher MCP-1 concentrations compared to pGlu ¹ -MCP-1 (FIG. 23). Pre-incubation of 3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea with Gln ¹ -MCP-1 prevents pGlu- formation and thus prevents DP4 cleavage It gives a peptide that is fragile. Thus, inhibition of QC causes MCP-1 N-terminal destabilization via degradation by DP4 and thus its inactivation with respect to monocyte chemotaxis activity.

  In addition, the ability of MCP-2, MCP-3, and MCP-4 with N-terminal glutamine or pyroglutamic acid to attract human THP-1 monocytes was investigated. Like MCP-1, pGlu-formation at the N-terminus of MCP-2 and MCP-3 has no effect on performance compared to the respective glutamine-precursor. However, for MCP-4, pGlu-formation slightly increases the ability of the peptide (FIG. 24). However, since glutaminyl-precursors are cleaved by DP4 (Figures 19, 20, 21), they also run the ability of NCP cleaved DP4 cleavage products of MCP-2, MCP-3, and MCP-4, Investigated using a chemical assay. For all three variants, cleavage by two amino acids causes partial inactivation of chemokines (Figure 25). Thus, pGlu- formation at the N-terminus of all known MCPs not only protects against N-terminal truncation, but also protects against loss of chemotaxis performance. Thus, the proposed approach to reduce the activity of MCP-1 by inhibiting N-terminal maturation applies to all members of the MCP family in humans.

Application of QC inhibitors to a model of LPS-induced sepsis in rats General 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride In order to investigate anti-inflammatory properties, inhibitors were applied to a model of LPS-induced sepsis in rats. As a marker of the onset of inflammatory response, the level of cytokine TNFα was determined in response to QC inhibitor treatment. As illustrated in FIG. 26, the application of 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea hydrochloride is low dose (5 mg / kg) Causes a dose-dependent decrease in TNFα levels ranging from 1 to intermediate doses (20 mg / kg). In addition, the highest dose (80 mg / kg) also reduced TNFα- levels in plasma, but a slight increase was observed compared to the intermediate dose. Thus, QC inhibitor application can significantly reduce the inflammatory response shown herein, taking TNFα as an example. In experiments, the effects of QC inhibitors are highly specific due to MCP N-terminal destabilization, but this inactivation of chemokines also affects other inflammatory parameters such as TNFα. It shows having. Thus, suppression of other pro-inflammatory cytokines is a further result of the proposed concept that destabilizes MCP. This approach is therefore suitable for developing pharmacotherapy for different inflammatory disorders that alter the degree of MCP action.

Application of QC inhibitors to a model of thioglycolate-induced peritonitis in mice To further investigate the effects of QC inhibitor administration on immune cell migration in vivo, 1- (3- (1H-imidazole hydrochloride) 1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea was applied to a model of peritonitis induced by thioglycolate in mice. The cellular composition of the peritoneal dialysate was determined with particular emphasis on infiltrating monocytes at 4 and 24 hours after thioglycolic acid induction. As shown in FIG. 27, the QC inhibitor 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea was dose-dependent after 4 hours. The number of monocytes that infiltrate the peritoneum was reduced. In addition, the presence of Moma2-positive monocytes / macrophages was assessed 24 hours after thioglycolate application. As shown in FIG. 28, the QC inhibitor 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea is significantly Moma2-positive The number of cells was also reduced. Thus, inhibition of QC destabilizes the N-terminus of MCP in vivo.

  This experiment demonstrates the applicability of MCP destabilization by QC inhibition to observe therapeutic effects. Some inflammatory disorders, such as but not limited to atherosclerosis, and monocyte recruitment, a common feature of restenosis, are suppressed. Thus, this experiment provides a method for characterizing QC inhibitors for their applicability in different inflammatory disorders.

Table 1: Primers used

Table 2: Dosing of QC inhibitors in sepsis induced by LPS in rats

(Synthesis of QC inhibitors)
(Synthesis Scheme 1): Synthesis of Examples 1-53, 96-102, and 136-137

Reagents and conditions: (a) NaH, DMF, 4 hours, room temperature; (b) 8 hours, 100 ° C .; (c) H ₂ N—NH ₂ , EtOH, 8 hours, reflux, then 4N HCl, 6 hours, Reflux, (d) R ³ —NCO, EtOH, 6 h, reflux, (e) 3,4 dimethoxy-phenyl-isothiocyanate.

(Synthesis Scheme 2): Synthesis of Examples 54-95

  Reagents and conditions: (a) R-NCS, EtOH, 6 hours, reflux; (b) WSCD, 1H-imidazole-1-propanamine, DMF, 2 hours, room temperature.

(Synthesis Scheme 3): Synthesis of Examples 103-105

Reagents and conditions: (a) NaH, DMF, room temperature, 3 hours; (b) LiAlH ₄ , dioxane, reflux, 1 hour; (c) R-NCS, EtOH, reflux, 6 hours.

試薬、及び条件：（a）EtOH、2時間、還流。 (Synthesis Scheme 4): Synthesis of Examples 106-109

Reagents and conditions: (a) EtOH, 2 hours, reflux.

(Synthesis Scheme 5): Synthesis of Examples 110-112

  Reagents and conditions: (a) 1H-imidazole-1-propanamine, triethylamine, toluene, reflux for 12 hours.

(Synthesis Scheme 6): Synthesis of Examples 113-132

  Reagents and conditions: (a) CAIBE, 1H-imidazole-1-propanamine, dioxane, 0 ° C., 12 hours; (b) Laweson reagent, EtOH, reflux, 8 hours.

(Synthesis Scheme 7): Synthesis of Examples 133-135

Reagents and conditions: (a) 1H-imidazole-1-propane acid chloride, CH ₂ Cl ₂ , −10 ° C., 1 hour; (b) Lawesson reagent, dioxane, reflux, 8 hours.

(Synthesis Scheme 8): Synthesis of Example 138

  Reagents and conditions: (a) EtOH, reflux, 8 hours.

(Synthesis Scheme 9): Synthesis of Example 139

Reagents and conditions: (a) 75% concentrated H ₂ SO ₄ , 4 hours.

(Synthesis Scheme 10): Synthesis of Example 140

  Reagents and conditions: (a) acetonitrile, reflux, 2 hours.

(Synthesis Scheme 11): Synthesis of Example 141

Reagents and conditions: (a) NaH, DMF, 4 hours, room temperature; (b) 8 hours, 100 ℃; (c) H 2 N-NH 2, EtOH, 8 hours, under reflux, then 4N HCl, 6 hours, Reflux, (d) 3,4 dimethoxy-phenyl-isothiocyanate, EtOH, reflux for 6 hours.

(Analysis conditions)
ESI-Mass spectra were obtained with a SCIEX API 365 spectrometer (Perkin Elmer). ¹ H-NMR (500MHz) data were recorded on a BRUKER AC 500 using DMSO-D ₆ as solvent. Chemical shifts are expressed as parts per million per low magnetic field from tetramethylsilane. The split patterns were named as follows: s (singlet), d (doublet), dd (doublet of doublet), t (triplet), m (multiplet), and br (Broad signal).

(Detailed synthesis description).
Examples 1-12 and 14-53
1H-imidazole-1-propanamine was reacted with the corresponding isothiocyanate in ethanol at reflux for 8 hours. Thereafter, the solvent was removed and the remaining oil was dissolved in methylene chloride. The organic layer was washed twice with a saturated solution of NaHCO ₃ followed by NaHSO ₄ and brine, dried and then evaporated. The remaining solid was recrystallized from ethyl acetate to give the example thiourea in 80-98% yield.

Example 13
1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) thiourea
4.0 mmol 3,4-dimethoxyphenyl isothiocyanate and 4.0 mmol 3- (1H-imidazol-1-yl) alkyl-1-amine were dissolved in 10 mL absolute ethanol. After stirring for 2 hours under reflux, the solvent was evaporated and the resulting solid was recrystallized from ethanol.

。 Yield: 0.66 g (51.3%); mp: 160.0-161.0 ° C

.

Examples 96-102
1H-imidazole-1-propanamine was reacted with the corresponding isocyanate in ethanol under reflux for 8 hours. Thereafter, the solvent was removed and the remaining oil was dissolved in methylene chloride. The organic layer was washed twice with a saturated solution of NaHCO ₃ followed by NaHSO ₄ and brine, dried and then evaporated. The remaining solid was recrystallized from ethyl acetate to give the example urea in 85-90% yield.

Examples 136, 137
1H-imidazole-1-alkylamine was prepared according to the literature from -bromo-alkyl-phthalimide, and imidazolium salts, followed by hydrazine decomposition. The resulting product was converted to thiourea according to Example 1-53 to give yields of 88% (Example 136) and 95% (Example 137).

Examples 54-95
All examples were made from the corresponding thiourea by reacting with water-soluble carbodiimide (WSCD) and 1H-imidazole-1-propanamine in dry dimethylformamide for 2 hours at room temperature. Trisubstituted guanidine was obtained in a yield of%.

Examples 103-105
Imidazole was reacted with the corresponding bromomethylphenyl cyanide in DMF using 1 equivalent of NaH for 3 hours at room temperature to give 1H-imidazole-1-methylphenyl cyanide. The solvent was removed and the resulting oil was redissolved in dioxane. The cyanide was converted to the corresponding amine using 1 equivalent of LiAlH ₄ . After adding a saturated solution of KHSO ₄ , the dioxane was evaporated and the aqueous layer was extracted with CHCl ₃ . The organic layer is concentrated in vacuo to convert the amine to the corresponding thiourea according to Examples 1-53, 78% (Example 103), and 65% (Example 104), and 81% (Example 105). ) Was given.

Examples 106-109
Starting from the corresponding methanesulfonate-2-methylpropyl-phthalimide, the amine was synthesized as described for the amine of Examples 136-137. The resulting product was converted to thiourea according to Example 1-53 to give Examples 106-109 in 25-30% overall yield.

Examples 110-112
1H-imidazole-1-propanamine was reacted with the corresponding 2-chlorobenzo [d] thiazole in toluene for 24 hours at a temperature of 130 ° C. After removing the solvent and recrystallization from methanol, Examples 110-112 were obtained in amounts of 55-65%.

Examples 113-118, 120-124, and 126-132
1H-imidazole-1-propanamine was reacted with the corresponding 2-phenylacetic acid in dry dioxane by adding 1 equivalent of CAIBE and N-methylmorpholine at a temperature of 0 ° C. After 2 hours, the mixture was warmed to room temperature and the mixture was stirred for 12 hours. After removal of the solvent, the resulting oil was redissolved in methylene chloride and the organic layer was washed with an aqueous solution of NaHCO ₃ and water, dried and the solvent was evaporated. The remaining oil was dissolved in dioxane and Laweson reagent was added. After stirring for 12 hours, a saturated solution of NaHCO ₃ was added. Dioxane was evaporated and the aqueous layer was extracted with ethyl acetate. The organic layer was separated, dried and the solvent was evaporated. The remaining solid was crystallized from acetyl acetate / ether to give 113-118, 120-124, and 126-132 in 62-85% overall yield.

。 Example 119
1 N- (3- (1H-imidazol-1-yl) propyl) -2- (3,4-dimethoxyphenyl) ethanethioamide
A mixture of 4.0 mmol triethylamine and 4.0 mmol 3- (1H-imidazol-1-yl) alkyl-1-amine, 20 mL dioxane was cooled to 4.0 mmol 2- (3,4-dimethoxy in 30 mL dioxane. To the stirred solution of phenyl) acetyl chloride was added dropwise. The mixture was warmed to room temperature and then stirred for 1 hour. After removing the solvent by reduced pressure, the residue was redissolved in 50 mL of dichloromethane. The organic layer was washed with NaHCO ₃ and a 30 mL saturated aqueous solution of water. The organic solution was dried, filtered and the solvent removed under pressure. After remelting in 50 mL dry dioxane, 2.2 mmol Lawesson reagent was added and the mixture was heated to 90 ° C. and stirred for 8 hours. The solvent was removed by reduced pressure and the residue was redissolved in 50 mL dichloromethane. The organic layer was washed ₃ times with a saturated aqueous solution of NaHCO ₃ followed by 3 times with water, dried, filtered and then the organic solvent was removed. The compounds were purified by chromatography using a 2 mm thick layer of silica plates and a centrifugal force-chromatography apparatus (Harrison Research Ltd.) utilizing a CHCl ₃ / MeOH gradient as the elution system.
Yield: 0.14 g (10.6%); Melting point: 148.0-150.0 ° C

.

Example 125
N- (3- (1H-imidazol-1-yl) propyl) -1- (3,4-dimethoxyphenyl) cyclopropanecarbothioamide
11.06 mmol 3,4-dimethoxyphenylacetonitrile, 34.8 mmol 2-bromo-1-chloroethanol, and 1.16 mmol triethylbenzylammonium hydrochloride were dissolved in 10 mL aqueous KOH (60%). The mixture was transferred to an ultrasonic bath and stirred vigorously at room temperature for 3 hours. The resulting suspension was diluted with 40 mL water and extracted three times with 20 mL dichloromethane. The combined organic layers were washed with aqueous hydrochloric acid (1N), dried over Na ₂ SO ₄ and the solvent removed under reduced pressure. The remaining oil was purified by flash chromatography using silica gel and ethyl acetate / heptane as the elution system to yield 0.81 g (34.4%) of 1- (3,4-dimethoxyphenyl) cyclopropanecarbonitrile.

3.9 mmol of 1- (3,4-dimethoxyphenyl) cyclopropanecarbonitrile and 11.2 mmol of KOH were suspended in 80 mL of ethylene glycol. The mixture was stirred at reflux for 12 hours. Then 80 mL of water was added and the aqueous layer was extracted twice with ether. After adjusting the pH to a value of pH = 4-5 using HCl (1N), the aqueous layer is extracted three times with ether, then the combined organic layers are dried over Na ₂ SO ₄ to remove the solvent This yielded 0.81 g (93.5%) of 1- (3,4-dimethoxyphenyl) cyclopropanecarboxylic acid.

3.44 mmol 1- (3,4-dimethoxyphenyl) cyclopropanecarboxylic acid, 3.5 mmol N-methylmorpholine and 3.5 mmol isobutylchloroformiat dissolved in dry tetrahydrofuran at -15 ° C Stir for 15 minutes. Then 3.5 mmol of 3- (1H-imidazol-1-yl) alkyl-1-amine was added and the mixture was warmed to 0 ° C. and stirred for 12 hours. The solvent was removed under reduced pressure and the remaining oil was redissolved in chloroform. The organic layer was then washed twice with a saturated aqueous solution of NaHCO ₃ and then dried over Na ₂ SO ₄ to remove the solvent. Purification was carried out by forced chromatography by centrifugation using a 2 mm thick layer of silica plates and a Chromatotron® apparatus (Harrison Research Ltd.) utilizing a CHCl ₃ / MeOH gradient as elution system. 0.671 g (59.3%) of N- (3- (1H-imidazol-1-yl) propyl) -1- (3,4-dimethoxyphenyl) cyclopropane-carboxamide.

。 After remelting in 30 mL dry dioxane, 1.43 mmol Lawesson reagent was added and the mixture was heated to 90 ° C. and stirred for 8 hours. The solvent was removed by reduced pressure and the remaining residue was dissolved in 50 mL of dichloromethane. The organic layer was washed ₃ times with a saturated aqueous solution of NaHCO ₃ followed by 3 times with water, dried, filtered and then the organic solvent was removed. The compounds were purified by chromatography using a 2 mm thick layer of silica plates and a centrifugal-chromatographic apparatus (Harrison Research Ltd.) utilizing a CHCl ₃ / MeOH gradient as the elution system.
Yield: 0.33 g (46.2%); Melting point: 127.0-127.5 ° C

.

Examples 133-135
A mixture of 1 equivalent of triethylamine and 3,4-dimethoxyaniline in dioxane was added to a stirred solution of the corresponding ω-bromoalkyl acid chloride at a temperature of 0 ° C. The solution was warmed to room temperature and stirred for 2 hours. The solvent was evaporated and the remaining oil was redissolved in dichloromethane. The organic layer was washed with water, dried, filtered and the solvent removed under reduced pressure.

  Imidazole and sodium hydride were suspended in and the mixture was stirred for 3 hours at room temperature under inert conditions. ω-Bromo-N- (3,4-dimethoxy-phenyl) alkylamide was added and the mixture was heated to 100 ° C. and stirred for 8 hours. The solvent was then evaporated, hot toluene was added and the solution was filtered. The solvent was then removed under reduced pressure. For example, as described in Examples 113-132, conversion to thioamide by Laweson's reagent was performed to give 133-135 in 13-20% overall yield.

  Analytical data for further examples synthesized according to the general synthetic scheme above is as follows:

。 Example 1: 1- (3- (1H-imidazol-1-yl) propyl) -3-methylthiourea Melting point: 122-122.5 ° C

.

。 Example 2: 1- (3- (1H-imidazol-1-yl) propyl) -3-tert-butylthiourea Melting point: 147.0-147.5 ° C

.

。 Example 3: 1- (3- (1H-imidazol-1-yl) propyl) -3-benzylthiourea Melting point: 127.0-128.0 ° C

.

。 Example 5: 1- (3- (1H-imidazol-1-yl) propyl) -3-phenylthiourea Melting point: 166.5-167.0 ° C

.

。 Example 6: 1- (3- (1H-imidazol-1-yl) propyl) -3- (4-fluorophenyl) thiourea Melting point: 147.0-148.0 ° C

.

。 Example 7: 1- (3- (1H-imidazol-1-yl) propyl) -3- (4-ethylphenyl) thiourea Melting point: 100.0-100.5 ° C

.

。 Example 8: 1- (3- (1H-imidazol-1-yl) propyl) -3- (4- (trifluoromethyl) phenyl) thiourea Melting point: 154.5-155.0 ° C

.

。 Example 10: 1- (3- (1H-imidazol-1-yl) propyl) -3- (4-acetylphenyl) thiourea Melting point: 170.0-171.0 ° C

.

。 Example 11: 1- (3- (1H-imidazol-1-yl) propyl) -3- (4-methoxyphenyl) thiourea Melting point: 125.0-125.5 ° C

.

。 Example 14: 1- (3- (1H-imidazol-1-yl) propyl) -3- (2,4-dimethoxyphenyl) thiourea Melting point: 120.0-120.5 ° C

.

。 Example 15: 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,5-dimethoxyphenyl) thiourea Melting point: 142.0-143.0 ° C

.

。 Example 23: 1- (3- (1H-imidazol-1-yl) propyl) -3- (2,3-dihydrobenzo [b] [1,4] dioxin-7-yl) -thiourea Melting point: 103.0 -103.5 ℃

.

。 Example 24: 1- (3- (1H-imidazol-1-yl) propyl) -3- (benzo [d] [1,3] dioxol-6-yl) thiourea Melting point: 115.0-115.6 ° C

.

。 Example 25: 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4,5-trimethoxyphenyl) thiourea Melting point: 124.5-125.5 ° C

.

。 Example 26: 1- (3- (1H-imidazol-1-yl) propyl) -3- (3-methoxyphenyl) thiourea Melting point: 89.5-90.0 ° C

.

。 Example 27: 1- (3- (1H-imidazol-1-yl) propyl) -3- (4-ethoxyphenyl) thiourea Melting point: 126.0-126.5 ° C

.

。 Example 33: 1- (3- (1H-imidazol-1-yl) propyl) -3- (4- (methylthio) phenyl) thiourea Melting point: 140.0-140.5 ° C

.

。 Example 42: 1- (3- (1H-imidazol-1-yl) propyl) -3- (4-nitrophenyl) thiourea Melting point: 165.0.166.0 ° C

.

。 Example 50: 1- (3- (1H-imidazol-1-yl) propyl) -3- (4- (dimethylamino) phenyl) thiourea Melting point: 146.5-147.0 ° C

.

。 Example 102: 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxyphenyl) urea Melting point: 114.5-115.0 ° C

.

。 Example 106: 1-((S) -3- (1H-imidazol-1-yl) -2-methylpropyl) -3- (3,4-dimethoxyphenyl) -thiourea Melting point: 150.5-151.5 ° C

.

。 Example 107: 1-((R) -3- (1H-imidazol-1-yl) -2-methylpropyl) -3- (3,4-dimethoxyphenyl) -thiourea Melting point: 155.0-157.5 ° C

.

。 Example 109: 1-((1-((1H-imidazol-1-yl) methyl) cyclopropyl) methyl) -3- (3,4-dimethoxy-phenyl) thiourea Melting point: 166.5-168.5 ° C

.

。 Example 110: N- (3- (1H-imidazol-1-yl) propyl) benzo [d] thiazol-2-amine

.

。 Example 111: N- (3- (1H-imidazol-1-yl) propyl) -6-chlorobenzo [d] thiazol-2-amine

.

。 Example 112: N- (3- (1H-imidazol-1-yl) propyl) -6-methoxybenzo [d] thiazol-2-amine

.

。 Example 115: (R) -N- (3- (1H-imidazol-1-yl) propyl) -2-phenylpropanethioamide Melting point: 82.0-82.5 ° C

.

。 Example 116: (S) -N- (3- (1H-imidazol-1-yl) propyl) -2-phenylpropanethioamide Melting point: 82.5-83.5 ° C

.

。 Example 121: N- (3- (1H-imidazol-1-yl) propyl) -1- (4-chlorophenyl) cyclobutanecarbo-thioamide Melting point: 137.5-139.0 ° C

.

。 Example 122: N- (3- (1H-imidazol-1-yl) propyl) -1- (4-chlorophenyl) cyclobutanecarbo-thioamide Melting point: 140.0-141.0 ° C

.

。 Example 123: N- (3- (1H-imidazol-1-yl) propyl) -1- (4-methoxyphenyl) cyclobutanecarbo-thioamide Melting point: 162.5-164.0 ° C

.

。 Example 124: N- (3- (1H-imidazol-1-yl) propyl) -1- (4-methoxyphenyl) cyclobutanecarb-bothioamide Melting point: 129.0-129.5 ° C

.

。 Example 134: 5- (1H-imidazol-1-yl) -N- (3,4-dimethoxyphenyl) pentanethioamide Melting point: 128.0-128.5 ° C

.

。 Example 136: 1- (2- (1H-imidazol-1-yl) ethyl) -3- (3,4-dimethoxyphenyl) thiourea Melting point: 157.5-159.0 ° C

.

Claims (32)

  A QC inhibitor for the treatment and / or prevention of inflammatory diseases or conditions selected from mild cognitive impairment (MCI), restenosis, and pancreatitis.   Use of a QC inhibitor for the treatment and / or prevention of an inflammatory disease or condition selected from mild cognitive impairment (MCI), restenosis, and pancreatitis.   Use of a QC inhibitor for the manufacture of a medicament for treating and / or preventing an inflammatory disease or condition selected from mild cognitive impairment (MCI), restenosis, and pancreatitis.   The QC inhibitor or use according to any one of claims 1 to 3, wherein the disease is mild cognitive impairment (MCI).   The QC inhibitor is a nootropic drug, neuroprotective drug, antiparkinsonian drug, amyloid protein deposition inhibitor, β amyloid synthesis inhibitor, antidepressant drug, anxiolytic drug, antipsychotic drug, and anti-multiple sclerosis drug The QC inhibitor according to any one of claims 1 to 4, or use, wherein the QC inhibitor is administered in combination with a further agent selected from the group consisting of:   The QC inhibitor or use according to any one of claims 1 to 3, wherein the disease is selected from restenosis and pancreatitis.   The QC inhibitor or use according to any one of claims 1 to 3, or 6, wherein the disease is restenosis.   The QC inhibitor is an angiotensin converting enzyme inhibitor (ACE); angiotensin II receptor blocker; diuretic; calcium channel blocker (CCB); β blocker; platelet aggregation inhibitor; cholesterol absorption modulator; A reductase inhibitor; compound that increases high density lipoprotein (HDL); renin inhibitor; IL-6 inhibitor; anti-inflammatory corticosteroid; antiproliferative drug; nitric oxide donor; inhibitor of extracellular matrix synthesis 8. A growth factor or cytokine signaling inhibitor; any one of claims 1-3, 6, or 7 administered in combination with an additional agent selected from the group consisting of an MCP-1 antagonist and a tyrosine kinase inhibitor; QC inhibitors or use.   A method of treating and / or preventing an inflammatory disease or condition selected from mild cognitive impairment (MCI), restenosis, and pancreatitis, wherein an effective amount of a QC inhibitor is administered to a subject in need thereof Said method.   10. The method of treatment and / or prevention according to claim 9, wherein the disease is mild cognitive impairment (MCI).   The QC inhibitor is a nootropic drug, neuroprotective drug, antiparkinsonian drug, amyloid protein deposition inhibitor, β amyloid synthesis inhibitor, antidepressant drug, anxiolytic drug, antipsychotic drug, and anti-multiple sclerosis drug. 11. The method of treatment and / or prevention according to claim 9 or 10, wherein the method is administered in combination with an additional agent selected from the group consisting of:   10. The method of treatment and / or prevention according to claim 9, wherein the disease is selected from restenosis and pancreatitis.   The method for treatment and / or prevention according to claim 9 or 12, wherein the disease is restenosis.   The method for treatment and / or prevention according to any one of claims 9 and 12, wherein the disease is pancreatitis.   The QC inhibitor is an angiotensin converting enzyme inhibitor (ACE); angiotensin II receptor blocker; diuretic; calcium channel blocker (CCB); β blocker; platelet aggregation inhibitor; cholesterol absorption modulator; A reductase inhibitor; compound that increases high density lipoprotein (HDL); renin inhibitor; IL-6 inhibitor; anti-inflammatory corticosteroid; antiproliferative drug; nitric oxide donor; inhibitor of extracellular matrix synthesis 15. A growth factor or cytokine signaling inhibitor; administered in combination with an additional agent selected from the group consisting of an MCP-1 antagonist and a tyrosine kinase inhibitor; Methods of treatment and / or prevention.   Use according to any of claims 2 to 8, wherein the disease and / or condition afflicts a human.   16. A method according to any of claims 9 to 15, wherein the disease and / or condition afflicts a human. 18. The QC inhibitor, use, or method according to any one of claims 1 to 17, wherein the QC inhibitor is a compound of formula 1 including pharmaceutically acceptable salts, solvates, and stereoisomers thereof. :

Where
A:
An alkyl chain, an alkenyl chain, or an alkynyl chain;
Or A:

Is a group selected from:
In the formula:
R ⁶ , R ⁷ , R ⁸ , R ⁹ , and R ¹⁰ are independently H, or an alkyl chain, alkenyl chain, alkynyl chain, cycloalkyl, carbocycle, aryl, heteroaryl, or heterocycle;
n and n ¹ are independently 1 to 5;
m is 1-5;
o is 0-4;
And B is:

A group selected from
In the formula:
D and E independently represent an alkyl chain, alkenyl chain, alkynyl chain, cycloalkyl, carbocycle, aryl, -alkylaryl, heteroaryl, -alkylheteroaryl, acyl, or heterocycle.
Z is CH or N;
X represents CR ²⁰ R ²¹ , O, S, NR ¹⁹ with the proviso that for formulas (VIII) and (IX), when Z = CH, X is O or S ;
R ¹⁹ is, H, alkyl, cycloalkyl, aryl, heteroaryl, - oxyalkyl, - oxyaryl, carbonyl, amido, hydroxy, selected from the group consisting of NO _2, NH _2, CN;
R ²⁰ and R ²¹ are independently selected from H, alkyl, cycloalkyl, heterocycle, aryl, heteroaryl, -oxyalkyl, -oxyaryl, carbonyl, amide, NO ₂ , NH ₂ , CN, CF ₃ Is;
X ¹ , X ² , and X ³ are independently O or S, provided that X ² and X ³ are not both O;
Y is O or S, provided that Y may not be O when the carbocyclic ring formed by R ¹⁷ and R ¹⁸ has three members of the ring;
Z is CH or N;
R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are independently H, alkyl chain, alkenyl chain, alkynyl chain, cycloalkyl, carbocycle, aryl, heteroaryl, heterocycle, halo, alkoxy-, -thioalkyl, May be selected from carboxyl, carboxylic ester, carbonyl, carbamide, carbimide, thiocarbamide, or thiocarbonyl, NH ₂ , NO ₂ ;
R ¹⁵ and R ¹⁶ are each independently H, or a branched or unbranched alkyl chain, or a branched or unbranched alkenyl chain;
R ¹⁷ and R ¹⁸ are independently selected from H or an alkyl chain, alkenyl chain, alkynyl chain, carbocycle, aryl, heteroaryl, heteroalkyl, or form a carbocycle up to 6 ring atoms Can be combined to
n is 0 or 1. The QC inhibitor, use, or method according to any one of claims 1 to 18, wherein the QC inhibitor, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof is selected from:
Compound of formula 1 *:

Or a compound of formula 1a,

Where R is defined in Examples 1 to 53:

A compound of formula 1b,

Wherein R ¹ and R ² are defined in Examples 54-95.

Or
A compound of formula 1c,

Wherein R ³ is defined in Examples 96-102.

Or
A compound of formula 1d,

Where the position on the ring is defined in Examples 103-105.
Or
Compound of formula 1e

Where R ⁴ and R ⁵ are defined in Examples 106-109.

Or
A compound of formula 1f,

Where R ⁶ is defined in Examples 110-112.

Or
A compound of formula 1g,

Wherein R ⁷ , R ⁸ , and R ⁹ are defined in Examples 113-132.

Or
A compound of formula 1h,

Where n is defined in Examples 133-135.

Or
A compound of formula 1i,

Where m is defined in Examples 136 and 137.

Or
A compound selected from Examples 138-141.

  20. The QC inhibitor is 1- (3- (1H-imidazol-1-yl) propyl) -3- (3,4-dimethoxy-phenyl) thiourea hydrochloride. A QC inhibitor, use, or method as described.   A diagnostic assay comprising a QC inhibitor. 24. The diagnostic assay of claim 21, wherein the QC inhibitor is a compound of formula 1 including pharmaceutically acceptable salts, solvates, and stereoisomers thereof:

In the formula:
A is: an alkyl chain, an alkenyl chain, or an alkynyl chain;
Or A is:

A group selected from
In the formula:
R ⁶ , R ⁷ , R ⁸ , R ⁹ , and R ¹⁰ are independently H, or an alkyl chain, alkenyl chain, alkynyl chain, cycloalkyl, carbocycle, aryl, heteroaryl, or heterocycle;
n and n ¹ are independently 1 to 5;
m is 1-5;
o is 0-4;
And B is a group selected from:

In the formula:
D and E independently represent an alkyl chain, alkenyl chain, alkynyl chain, cycloalkyl, carbocycle, aryl, -alkylaryl, heteroaryl, -alkylheteroaryl, acyl, or heterocycle.
Z is CH or N;
X represents CR ²⁰ R ²¹ , O, S, NR ¹⁹ with the proviso that for formulas (VIII) and (IX), when Z = CH, X is O or S ;
R ¹⁹ is, H, alkyl, cycloalkyl, aryl, heteroaryl, - oxyalkyl, - oxyaryl, carbonyl, amido, hydroxy, selected from the group consisting of NO _2, NH _2, CN;
R ²⁰ and R ²¹ are independently selected from H, alkyl, cycloalkyl, heterocycle, aryl, heteroaryl, -oxyalkyl, -oxyaryl, carbonyl, amide, NO ₂ , NH ₂ , CN, CF ₃ Is;
X ¹ , X ² , and X ³ are independently O or S, provided that X ² and X ³ are not both O;
Y is O or S, provided that Y may not be O when the carbocyclic ring formed by R ¹⁷ and R ¹⁸ has 3 members in the ring;
Z is CH or N;
R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are independently H, alkyl chain, alkenyl chain, alkynyl chain, cycloalkyl, carbocycle, aryl, heteroaryl, heterocycle, halo, alkoxy-, -thioalkyl, May be selected from carboxyl, carboxylic ester, carbonyl, carbamide, carbimide, thiocarbamide, or thiocarbonyl, NH ₂ , NO ₂ ;
R ¹⁵ and R ¹⁶ are each independently H, or a branched or unbranched alkyl chain, or a branched or unbranched alkenyl chain;
R ¹⁷ and R ¹⁸ are independently selected from H or an alkyl chain, alkenyl chain, alkynyl chain, carbocycle, aryl, heteroaryl, heteroalkyl, or form a carbocycle up to 6 ring atoms Can be combined to
n is 0 or 1. 23. The diagnostic assay of claim 21 or 22, wherein the QC inhibitor, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof is selected from:
Compound of formula 1 *:

Or
A compound of formula 1a,

Wherein R is defined in Examples 1-53:

A compound of formula 1b,

Wherein R ¹ and R ² are defined in Examples 54-95.

Or
A compound of formula 1c,

Wherein R ³ is defined in Examples 96-102.

Or a compound of formula 1d,

Where the position on the ring is defined in Examples 103-105,
Or
A compound of formula 1e,

Where R ⁴ and R ⁵ are defined in Examples 106-109.

Or
A compound of formula 1f,

Where R ⁶ is defined in Examples 110-112.

Or
A compound of formula 1g,

Wherein R ⁷ , R ⁸ , and R ⁹ are defined in Examples 113-132.

Or
A compound of formula 1h,

Where n is defined in Examples 133-135.

Or
A compound of formula 1i,

Where m is defined in Examples 136 and 137.

Or
A compound selected from Examples 138-141.

  24. The QC inhibitor according to any of claims 21 to 23, wherein the QC inhibitor is 1- (3- (H-imidazol-1-yl) propyl) -3- (3,4-dimethoxy-phenyl) thiourea hydrochloride. Diagnostic assay. A method for diagnosing any one of the diseases and / or conditions as defined in claim 1, comprising:
-Collecting a sample from a subject suspected of suffering from said disease and / or condition;
-Contacting said sample with a QC inhibitor; and
-Determining whether the subject is afflicted by the disease and / or condition;
Said method.   27. The method of claim 26, wherein the subject is a human. 28. The method of claim 26 or 27, wherein the QC inhibitor is a compound of formula 1, including pharmaceutically acceptable salts, solvates, and stereoisomers thereof:

In the formula:
A:
Is an alkyl chain, an alkenyl chain, or an alkynyl chain;
Or A is a group selected from:

In the formula:
R ⁶ , R ⁷ , R ⁸ , R ⁹ , and R ¹⁰ are independently H, or an alkyl chain, alkenyl chain, alkynyl chain, cycloalkyl, carbocycle, aryl, heteroaryl, or heterocycle;
n and n ¹ are independently 1 to 5;
m is 1-5;
o is 0-4;
And B is a group selected from:

In the formula:
D and E independently represent an alkyl chain, alkenyl chain, alkynyl chain, cycloalkyl, carbocycle, aryl, -alkylaryl, heteroaryl, -alkylheteroaryl, acyl, or heterocycle.
Z is CH or N;
X represents CR ²⁰ R ²¹ , O, S, NR ¹⁹ with the proviso that for formulas (VIII) and (IX), when Z = CH, X is O or S ;
R ¹⁹ is, H, alkyl, cycloalkyl, aryl, heteroaryl, - oxyalkyl, - oxyaryl, carbonyl, amido, hydroxy, selected from the group consisting of NO _2, NH _2, CN;
R ²⁰ and R ²¹ are independently selected from H, alkyl, cycloalkyl, heterocycle, aryl, heteroaryl, -oxyalkyl, -oxyaryl, carbonyl, amide, NO ₂ , NH ₂ , CN, CF ₃ Is;
X ¹ , X ² , and X ³ are independently O or S, provided that X ² and X ³ are not both O;
Y is O or S, provided that Y may not be O when the carbocyclic ring formed by R ¹⁷ and R ¹⁸ has 3 members in the ring;
Z is CH or N;
R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are independently H, alkyl chain, alkenyl chain, alkynyl chain, cycloalkyl, carbocycle, aryl, heteroaryl, heterocycle, halo, alkoxy-, -thioalkyl, May be selected from carboxyl, carboxylic ester, carbonyl, carbamide, carbimide, thiocarbamide, or thiocarbonyl, NH ₂ , NO ₂ ;
R ¹⁵ and R ¹⁶ are each independently H, or a branched or unbranched alkyl chain, or a branched or unbranched alkenyl chain;
R ¹⁷ and R ¹⁸ are independently selected from H or an alkyl chain, alkenyl chain, alkynyl chain, carbocycle, aryl, heteroaryl, heteroalkyl, or form a carbocycle up to 6 ring atoms Can be combined to
n is 0 or 1. 28. The method of any one of claims 25 to 27, wherein the QC inhibitor, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, is selected from:

Or
A compound of formula 1a,

Wherein R is defined in Examples 1-53:

Or
A compound of formula 1b,

Wherein R ¹ and R ² are defined in Examples 54-95.

Or
A compound of formula 1c,

Wherein R ³ is defined in Examples 96-102.

Or a compound of formula 1d,

Where the position on the ring is defined in Examples 103-105,
Or
A compound of formula 1e,

Where R ⁴ and R ⁵ are defined in Examples 106-109.

Or
A compound of formula 1f,

Where R ⁶ is defined in Examples 110-112.

Or
A compound of formula 1g,

Wherein R ⁷ , R ⁸ , and R ⁹ are defined in Examples 113-132.

Or
A compound of formula 1h,

Where n is defined in Examples 133-135.

Or
A compound of formula 1i,

Where m is defined in Examples 136 and 137.

Or a compound selected from Examples 138-141.

  29. The QC inhibitor according to any one of claims 25 to 28, wherein the QC inhibitor is 1- (3- (H-imidazol-1-yl) propyl) -3- (3,4-dimethoxy-phenyl) thiourea hydrochloride. Method.   30. The method of any of claims 25-29, wherein the sample is a blood sample, a serum sample, a cerebrospinal solution sample, or a urine sample.   A diagnostic kit for performing the method of claims 25 to 30, comprising the diagnostic assay according to any of claims 21 to 24 as a detection means, and a determination means.   A pharmaceutical composition comprising the QC inhibitor according to any one of claims 1, 4 to 6, or 18 to 20.

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